Aptamer-modified polymeric materials for the binding of factors in a wound environment

ABSTRACT

Aptamer-modified polymers and materials thereof, used for binding factors in a wound bed. The aptamer-modified materials can be polypeptides conjugated to polymer foam materials and used, for example, as dressings, wound inserts, or pads.

The present application is a continuation of U.S. application Ser. No.13/458,071 filed Apr. 27, 2012, which claims priority to U.S.Provisional Application Ser. No. 61/518,148 filed Apr. 29, 2011. Theentire content of each of the above disclosures is incorporated hereinby reference.

BACKGROUND

1. Field of the Invention

The present disclosure relates generally to healing of wounds andwound-treatment therapies. More particularly, but not by way oflimitation, the present disclosure relates to modified materials, forexample, silyl modified polyurethane foam for the reversible binding offactors in a wound bed.

2. Background Information

Clinical studies and practice have shown that providing a reducedpressure in proximity to a tissue site augments and accelerates thegrowth of new tissue at the tissue site. The applications of thisphenomenon are numerous, but application of reduced pressure has beenparticularly successful in treating wounds. This treatment (frequentlyreferred to in the medical community as “negative pressure woundtherapy,” “reduced pressure therapy,” or “vacuum therapy”) provides anumber of benefits, including faster healing and increased formulationof granulation tissue. One of the major clinical benefits of negativepressure wound therapy is its ability to effectively eliminate woundexudate, thereby reducing edema and allowing tissue decompression.Negative pressure wound therapy may not always be able to differentiatebetween harmful and beneficial factors removed from the wound. Coatingused to address this such as collagen, PVA, PEG, and fibrinogen oftensuffer from not being covalent, uniform, or target specific. Typicallythey cannot bind proteins at a specific site and present them to cellsin a manner that allows the active site to retain its function.Improvements that would allow the binding of molecules in a covalentmanner, specify the types of compounds that could be bound, the chemicalreactions with which to bind them, and/or the orientation with which theprotein is presented to the cells, e.g., for use in a dressing, woundinsert, pad, etc., would therefore be highly desirable.

SUMMARY

The present disclosure provides novel materials, including aptamer(e.g., polypeptide) modified polymers, which may be used for the bindingof factors, such as from a wound bed. In some embodiments, the factorsare endogenous. In others they are exogenous. In some embodiments, thebind is reversible, in others it is irreversible.

In some embodiments a polypeptide is provided comprising an amino acidsequence identical to SEQ ID NO: 2 (P16; GHQGQHIGQMS), a sequence atleast 90% identical to SEQ ID NO: 2 or a sequence comprising 1, 2, or 3amino acid substitutions or deletions relative to SEQ ID NO: 2. In someaspects, the amino acid sequence further comprises an amino or carboxylterminal Cys residue (e.g., the amino or carboxyl terminus of apolypeptide of the embodiments comprises a PEG spacer-cysteine residue).For example, the amino acid sequence can comprise the sequenceAEEAc-Cys-NH₂ at the C-terminus. In still further aspects, a wounddressing is provided comprising a polymer foam substrate conjugated to apolypeptide of the embodiments, such as a polypeptide comprising anamino acid sequence of SEQ ID NO: 2 (GHQGQHIGQMS) or a sequence at least90% identical to SEQ ID NO: 2.

In certain embodiments a wound dressing is provided comprising a polymerfoam substrate conjugated to a polypeptide, wherein the polypeptidecomprises an amino acid sequence identical to SEQ ID NO: 1 (P22;NAIQEARRLLNLSRD), a sequence at least 90% identical to SEQ ID NO:1 or asequence comprising 1, 2, or 3 amino acid substitutions or deletionsrelative to SEQ ID NO: 1. In some aspects, the amino acid sequencefurther comprises an amino or carboxyl terminal Cys residue (e.g., theamino or carboxyl terminus of a polypeptide of the embodiments comprisesa PEG spacer-cysteine residue). For example, the amino acid sequence cancomprise the sequence AEEAc-Cys-NH₂ at the C-terminus.

In some embodiments, a polypeptide of the embodiments is covalentlyattached to a polymer foam (e.g., to form a wound dressing). In someaspects, a polypeptide is attached to the polymer foam through athioether linker. For example, the linker can be an EMCS-derived linkeror a sulfo-EMCS-derived linker. In some aspects, the thioether linkercomprises a Cys residue in the polypeptide, such as a Cys residue in aAEEAc-Cys-NH₂ sequence of the polypeptide. In still further aspects, alinker between a polypeptide and a polymer foam can be a silyl derivedlinker (e.g., a substituted silyl derived linker derived fromaminoundecyltriethoxysilane or aminopropyldiisopropylethoxysilane). Instill further aspects, a polypeptide of the embodiments is attached to apolymer foam via a non-covalent binding, such as by a biotin-avidinbinding.

In certain aspects, a wound dressing of the embodiments furthercomprises a growth factor, chemokine or cytokine bound to the wounddressing (e.g., bound to the wound dressing via an interaction with apolypeptide of the embodiments). In some aspects, the growth factor isgranulocyte macrophage colony stimulating factor (GM-CSF). In certainaspects, the growth factor is vascular endothelial growth factor (VEGF).

In some embodiments, a polypeptide of the embodiments is attached to apolymer foam to form a wound dressing. For example, the polymer foamsubstrate can be a reticulated open-celled foam. Examples of polymerfoams include, without limitation, foam substrates comprising polyvinylalcohol, polyurethane, polypropylene, polystyrene, polyols, poloxamer,aminoglycosides, amino sugars, or a combination of one or more of these.

In further embodiments a method is provided for binding a growth factor,chemokine or cytokine comprising contacting a fluid (such as a bodyfluid) comprising the growth factor, chemokine or cytokine with a wounddressing according to embodiments, thereby binding the growth factor,chemokine or cytokine. In some aspects, a wound dressing of theembodiments is adapted for using in applying negative pressure to awound site. In further aspects, a method comprises removing some or allof the fluid in contact with the wound dressing. In still furtheraspects, a method of the embodiments further comprises contacting thewound dressing with at least a second fluid wherein some or all of thegrowth factor, chemokine or cytokine bound to the wound dressing iseluted into the second fluid. In certain aspects, a method of theembodiments is defined as an in vitro method.

In yet a further embodiment, a composition is provided for the treatmentof a wound, comprising a wound dressing according to the embodiments. Insome aspects, a method is provided for treating a wound comprisingcontacting a wound site with a wound dressing of the embodiments. Incertain aspects, a method of the embodiments further comprises applyinga negative pressure to the wound site.

Any embodiment of any of the present systems and/or methods can consistof or consist essentially of—rather thancomprise/include/contain/have—any of the described steps, elements,and/or features. Thus, in any of the claims, the term “consisting of” or“consisting essentially of” can be substituted for any of the open-endedlinking verbs recited above, in order to change the scope of a givenclaim from what it would otherwise be using the open-ended linking verb.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings illustrate by way of example and not limitation.For the sake of brevity and clarity, every feature of a given structureis not always labeled in every figure in which that structure appears.Identical reference numbers do not necessarily indicate an identicalstructure. Rather, the same reference number may be used to indicate asimilar feature or a feature with similar functionality, as maynon-identical reference numbers.

FIG. 1 depicts a side view of one embodiment of the present wounddressings having one of the present modified wound inserts and coupledto a wound site and to a wound treatment apparatus.

FIG. 2 depicts an enlarged side view of the modified wound insert ofFIG. 2.

FIG. 3 depicts a schematic block diagram of one embodiment of a woundtreatment apparatus that can comprise and/or be coupled to and/or beused with the present wound dressings and/or modified wound inserts.

FIG. 4 depicts a schematic diagram according to some embodiments of thepresent invention. In this side view, a polyurethane-based polymerhaving free hydroxy groups is modified with a R′—(CH₂)_(n)—Si(OH)₃reagent.

FIG. 5 depicts results comparing the number of free amino groups ofsilylated 30 phr chitosan foam (“30 PHR Si”) with silylated reticulatedopen cell foam (“ROCF”) using an o-phtaldialdehyde (OPA) assay. In bothcases the silylation group was (H₂N(CH₂)₃)(i-Pr)₂Si— from the compound602 (aminopropyldiisopropylethoxysilane). The results are provided interms of relative fluorescence units (RFU). OPA alone, which is asensitive detection reagent for amines, also serves as the control.

FIG. 6 depicts results comparing the relative fluorescence units(“RFUs”) of reticulated open cell foam that was silylated with the group(H₂N(CH₂)₁₁)(EtO)₂Si— from compound 630 (aminoundecyltriethoxysilane)and treated with Texas Red® with non-silylated reticulated open cellfoam (“ROCF”), alone and together with Texas Red®. The results areprovided in terms of relative fluorescence units (RFU). PBS, and ROCF inPBS were also analyzed to ensure that autofluorescence of thesematerials would not skew RFU readings. PBS is phosphate buffered saline.

FIG. 7 depicts three repeat units of a polyurethane based on thepolymerization of toluene diisocyanate (TDI), chitosan, and Voranol™3010 triol.

FIG. 8: Silylated with compound 602, 30 PHR Chitosan/P-22 captures moregranulocyte macrophage colony stimulating factor (GM-CSF). Briefly, 333ng/mL of GM-CSF was incubated with the samples for 24 hr. The unboundprotein was washed off then the samples were eluted with animidazole-based buffer, E1. The samples were cleaned up via bufferexchange with PBS and quantified by ELISA. The results indicate that thepositive sample with P-22 captured 299 pg/mL of GM-CSF, whereas the 30PHR alone bound 124 pg/mL.

FIGS. 9A & B provide images of silanated ROCF with Texas Red fluorescentdye. FIG. 9A shows a Brightfield image showing the orientation andappearance of -Sil/ROCF in regular light. FIG. 9B shows TRITC imaging ofTexas Red conjugated to 630 showing successful conjugation of the dye tothe -Sil. This provides evidence that 630 was successfully deposited onthe ROCF. Both images were taken at 20× magnification.

FIG. 10 provides structure and connectivity information for a portion ofan aptamer-modified polymeric foam embodiment comprising an oligopeptide(P16; SEQ ID NO: 2), an AEEAc-Cys linker, an EMCS linker, a substitutedsilyl group, and a polyurethane backbone based on a PEGylated triol anda toluene diisocyanate.

FIG. 11 provides elution results for the P22/GM-CSF experiment. P22 wasobtained from the Bachem Americas Inc. 2010 peptide catalog. P22 is aGM-CSF antagonist that binds GM-CSF in an inhibitory manner. Briefly,330 nmol P22 was conjugated to a polyurethane-based ROCF via Sulfo-EMCSchemistry. The P22-linker-foam construct was incubated with 1 μg GM-CSFovernight, washed five times, and subjected to stringent elutions. Thewash preceding the elutions showed that non-specifically bound proteinwas reduced to negligible levels (<100 pg/mL). Upon elution, the samplewith the most capture peptide, S3, outperformed ROCF alone by 49%. Thetotal amount of GM-CSF bound to ROCF/P22, 608 pg/mL, was greater thanthe amount of GM-CSF bound non-specifically to the negative control, C1,376 pg/mL. Samples S1-S3 all contained peptide-linker-foam withSulfo-EMCS concentration increasing from 51 to S3 (51=10×Sulfo-EMCS+P22; S2=25× Sulfo-EMCS+P22 and S3=50× Sulfo-EMCS+P22), C1 wasROCF alone, C2 was Silanated ROCF+EMCS−P22 and C3 was SilanatedROCF−EMCS+P22. There were no positive controls available for thisexperiment, as no technology has yet been proven to covalently conjugatepeptides to polyurethane. However, positive controls were laterdeveloped for subsequent experiments based on experimental results. Dataare given as mean±standard deviation.

FIG. 12 provides structure and connectivity information for a portion ofan aptamer-modified polymeric foam embodiment comprising an oligopeptide(P16; SEQ ID NO: 2), an AEEAc-Cys linker, an EMCS linker, a substitutedsilyl group, and a polyurethane-copolymer based on a PEGylated triol, atoluene diisocyanate and a chitosan oligomer.

FIG. 13 provides structure and connectivity information for a portion ofan aptamer-modified polymeric foam embodiment comprising an oligopeptide(P16; SEQ ID NO: 2), an AEEAc-Cys linker, an EMCS linker, and apolyurethane-copolymer based on a PEGylated triol, a toluenediisocyanate and a chitosan oligomer.

FIG. 14 shows that ROCF captures 82% more VEGF with P16 (a de novodesigned capture peptide for VEGF) covalently bound to it. C1 was ROCFalone; C2 was ROCF+EMCS−P16; C3 was ROCF−EMCS+P16; C4 was silanated ROCFalone; C5=silanated ROCF+EMCS−P16; C6 was silanated ROCF−EMCS+P16; S1was 50× Sulfo-EMCS+P16; and S2 was 100× Sulfo-EMCS+P16. W=Finalsubsequent PBS wash. E1=50 mM Tris, 80 mM NaCl, 250 mM Imidazole, 45min. W5 (left) shows VEGF (pg/mL) non-specifically retained in the ROCFin the final PBS wash. E1 (right) indicates the amount of VEGF dislodgedfrom the foam and peptide with an organic solvent after the last wash.The elution data indicate that S2 (P16-linker-foam) bound 82% more VEGFthan C4 (silanated ROCF), the negative control with the most abundantbound VEGF. S2 bound 584 pg/mL, whereas C4 bound 321 pg/m. S2 has thehighest concentration of EMCS and therefore the most covalently boundP16. This data shows the strong correlation between the amount of P16covalently linked to the foam and the amount of target protein caught bythe foam. Data are given as mean±standard deviation.

FIG. 15 shows an aptamer modified polymer used in a dressing used inconjunction with negative pressure wound therapy (NPWT). In thisembodiment, the aptamer is selective for MCP1, a chemotactic molecule tomacrophages. Such a modified dressing may be used to stimulatemacrophage migration into the wound and thereby progress the wound froma chronic to a healing state.

FIG. 16 provides structure and connectivity information for a portion ofan aptamer-modified polymeric foam embodiment comprising an oligopeptide(P16; SEQ ID NO: 2) covalently attached to a polyurethane-copolymerbased on a PEGylated triol, a toluene diisocyanate and a chitosanoligomer.

FIG. 17 depicts a schematic diagram according to some of the embodimentsof the present invention. In this side view, an insert modified withspecifically designed proteins is shown binding with differentdissociation constants (K_(d) values) to different factors. In thisembodiment, the modified insert is being flushed (continuously orintermittently) with a treatment solution (metered bolus) containing avariety of agents, for example, agents for wound cleaning, promotinghealing, solution stability, etc. The block arrow labeled “Exudate”represents mixture leaving the material, including treatment solutionand exudate from a wound that dressing may be in contact with.

FIG. 18—VEGF in solution was passed over beads linked to an anti-VEGFantibody. After passing the solution over the beads, beads were spundown and washed. The washed beads were then used for endothelial cellmigration assays. Results from this experiment showed that after 3hours, 2 fold more cells had migrated towards the beads bound to theantibody (and VEGF) than beads with no antibody.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In some aspects, the present disclosure provides aptamer modifiedmaterials, which may be used, in some embodiments, as wound inserts forcatching and concentrating specific factors in the wound environment,for example, factors triggering a biological response such asangiogenesis, or to deliver biomolecules or bioactive compounds to awound bed. In some embodiments, the factors are endogenous. In othersthey are exogenous. In some embodiments the bind is reversible, inothers it is irreversible.

In one aspect, the invention provides aptamer-functionalized wounddressings, comprising: (a) a substrate; and (b) an aptamer covalentlyattached to the polymer foam. In some embodiments, the substrate is apolymer foam. In some embodiments, the aptamer comprises a apolypeptide, a small-molecule binder, a dendrimer, a nanoparticle,and/or an oligonucleotide. In some embodiments, said aptamer is apolypeptide. In some embodiments, the polypeptide is synthetic. In someembodiments, the polypeptide is natural.

In some embodiments, the polypeptide is covalently attached to thepolymer foam. In some embodiments, C-terminus of the polypeptidecomprises a PEG spacer-cysteine residue. In some embodiments, theaptamer is attached to the polymer foam through a linker. In someembodiments, the linker is an EMCS-derived linker or asulfo-EMCS-derived linker. In some embodiments, the linker is asubstituted silyl derived linker. In some embodiments, the substitutedsilyl derived linker is derived from compound 630(aminoundecyltriethoxysilane). In some embodiments, the substitutedsilyl derived linker is derived from compound 602(aminopropyldiisopropylethoxysilane). In some embodiments, the linkercomprises a first and second linker, wherein the first linker is anEMCS-derived linker or a sulfo-EMCS-derived linker and the second linkeris a substituted silyl derived linker.

In some embodiments, the linker is bound to the polymer foam through anoxygen atom. In some embodiments, the linker is bound to the polymerfoam through a nitrogen atom. In some embodiments, the aptamer isconfigured to bind to endogenous or exogenous factors in a woundenvironment. In some embodiments, the binding is irreversible. In someembodiments, the binding is reversible. In some embodiments, the aptameris configured to bind to vascular endothelial growth factor. In someembodiments, the aptamer is configured to bind to platelet derivedgrowth factor. In some embodiments, the aptamer is configured to bind tofibroblast growth factor. In some embodiments, the aptamer is configuredto bind to keratinocyte growth factor. In some embodiments, the aptameris configured to bind to granulocyte macrophage colony stimulatingfactor.

In some embodiments, the substrate is a reticulated open-celled foam. Insome embodiments, the substrate comprises polyvinyl alcohol,polyurethane, polypropylene, polystyrene, polyols, poloxamer,aminoglycosides, amino sugars, or a combination of one or more of these.In some embodiments, the polymer foam comprises a first monomer subunit.In some embodiments, the first monomer subunit is a diisocyanate. Insome embodiments, the diisocyanate is toluene diisocyanate, methylenediphenyl diisocyanate, or ethyl lysine diisocyanate. In someembodiments, the polymer foam further comprises a second monomersubunit. In some embodiments, the second monomer is a triol. In someembodiments, the triol is a PEGylated glycerine. In some embodiments,the polymer foam further comprises a third monomer subunit. In someembodiments, the third monomer subunit is an aminoglycoside. In someembodiments, the aminoglycoside is chitosan. In some embodiments, theaminoglycoside is glucosamine. In some embodiments, the third monomersubunit is neomycin. In some embodiments, the third monomer subunit isdibekacin. In some embodiments, the third monomer subunit is kanamycin.In some embodiments, the third monomer subunit is tobramycin. In someembodiments, the third monomer subunit is streptomycin. In someembodiments, the third monomer subunit is gentamicin. In someembodiments, the polymer foam further comprises an oligomeric subunit.In some embodiments, the oligomeric subunit is a chitosan-basedoligomer. In some embodiments, the oligomeric subunit is aglucosamine-based oligomer.

In another aspect, the invention provides aptamer-modified polymers,wherein the polymer backbone comprises a first repeat unit of theformula:

wherein:

U1 is the unmodified portion of the first repeat unit;

L₁ is a linker molecule or a bond; and

L₂ is a linker molecule or a bond.

In some embodiments, L1 is a bond and the formula of the first repeatunit is further defined as:

In some embodiments, L2 is a bond and the formula of the first repeatunit is further defined as:

In some embodiments, L1 and L2 are bonds and the formula of the firstrepeat unit is further defined as:

In some embodiments, U1 is of the formula:

wherein the point of attachment of the side chain connects to L₁.

In some embodiments, U1 is of the formula:

wherein the point of attachment of the side chain connects to L₁.

In some embodiments, U1 is of the formula:

wherein the point of attachment of the side chain connects to L1.

In some embodiments, U1 is of the formula:

wherein:

n1, n2 and n3 are each independently from 0 to 20; and

the point of attachment of the side chain connects to L₁.

In some embodiments, U1 is based on a triol-based monomer. In someembodiments, the triol-based monomer is a PEGylated glycerine moleculehaving a molecular weight between 500 and 5,000 g/mole.

In some embodiments, L₁ is of the formula —O—Si(R₁)(R₂)—R₃—X—, wherein:

-   -   R₁ and R₂ are independently hydrogen, hydroxy, halo,        alkyl_((C≦6)) or alkoxy_((C≦6));    -   R₃ is alkanediyl_((C≦6)); and    -   X is —NH—, —C(O)— or —S—; and wherein X is connected to L₂ or to        the aptamer.

In some embodiments, R₁ and R₂ are isopropyl. In some embodiments, R₁and R₂ are hydroxy. In some embodiments, R₃ is alkanediyl_((C3-11)). Insome embodiments, R₃ is —(CH₂)₃—. In some embodiments, R₃ is —(CH₂)₁₁—.In some embodiments, X is —NH—.

In some embodiments, L₁ or L₂ is an amino thio linker. In someembodiments, L₁ or L₂ is an NHS-Maleimide crosslinker. In someembodiments, L₁ or L₂ is an NHS-Haloacetyl crosslinker. In someembodiments, L₁ or L₂ is an NHS-Pyridyldithiol crosslinker.

In some embodiments, the amino thio linker is derived from a reagentselected from the group consisting of SM(PEG)24, SM(PEG)12, SM(PEG)8,SM(PEG)6, SM(PEG)4, Sulfo-LC-SMPT, SM(PEG)2, Sulfo-KMUS, LC-SMCC,LC-SPDP, Sulfo-LC-SPDP, SMPH, Sulfo-SMPB, SMPB, SMPT, STAB, Sulfo-SIAB,Sulfo-EMCS, EMCS, Sulfo-SMCC, SMCC, MBS, GMBS, Sulfo-GMBS, Sulfo-MBS,SPDP, SBAP, BMPS, AMAS, and SIA.

In some embodiments, the aptamer-modified polymer comprises a secondrepeat unit. In some embodiments, the second repeat unit is achitosan-based oligomer.

In some embodiments, U1 is of the formula:

wherein the point of attachment of the side chain connects to L1.

In some embodiments, L1 is of the formula:

In some embodiments, L2 is of the formula:

In some embodiments, L2 is of the formula —C(O)(CH2)5-S-Cys-AEEAc—,wherein AEEAc is:

In some embodiments, the aptamer-modified polymer further comprises asecond repeat unit.

In some embodiments, the second repeat unit has the formula:

In some embodiments, aptamer is an the oligopeptide having 8 to 12residues. In some embodiments, the oligopeptide has the sequenceGHQGQHIGQMS. In some embodiments, aptamer is P16. In some embodiments,aptamer is P22.

In some embodiments, the aptamer-modified polymer further comprises athird repeat unit of the formula:

wherein X is:

-   -   alkanediyl_((C≦12)) ⁻;    -   arenediyl_((≦12)) ⁻;    -   arenediyl_((≦6))—CH₂-arenediyl_((≦6)); or

substituted versions of any these groups.

In some embodiments, X is further defined as:

In some embodiments, the third repeat unit is a polyurethane repeatunit. In some embodiments, the third repeat unit is an ether-basedpolyurethane repeat unit. In some embodiments, the third repeat unit isan ester-based polyurethane repeat unit. In some embodiments, thepolymer is an open-celled foam.

In another aspect, the invention provides an aptamer-modified polymer orco-polymer comprising:

-   -   a) a first repeat unit having one or more hydroxy groups;    -   b) a second repeat using having one or more        —O—Si(R₁)(R₂)—R₃—X-linker-aptamer groups, wherein:        -   R₁ and R₂ are independently hydrogen, hydroxy, halo,            alkyl_((C≦6)) or alkoxy_((C≦6));        -   R₃ is alkanediyl_((C≦20));        -   X is —NH— or —S—;        -   the linker is —C(O)-alkanediyl_((C≦12)) ⁻; and        -   the aptamer is a biologically active protein that is bound            to the linker group through either an amino, a hydroxy or a            mercapto group.

In some embodiments, the first repeat unit has two hydroxy groups.

In some embodiments, the first repeat unit has the formula:

In some embodiments, R₁ and R₂ are isopropyl. In some embodiments,wherein R₁ and R₂ are hydroxy. In some embodiments, R₃ isalkanediyl_((C3-11)). In some embodiments, R₃ is —(CH₂)₃—.

In some embodiments, the second repeat unit has the formula:

In some embodiments, R₃ is —(CH₂)₁₁—. In some embodiments, the secondrepeat unit has the formula:

In some embodiments, X is —NH—. In some embodiments, the linker isfurther defined by the formula:

wherein the carbonyl carbon is attached to X.

In some embodiments, the aptamer is bound through the linker through themercapto group of a cysteine residue. In some embodiments, the aptameris granulocyte-macrophage colony stimulating factor (GM-CSF).

In some embodiments, the aptamer-modified polymer further comprises athird repeat unit of the formula:

wherein X is:

-   -   alkanediyl_((C≦12))—;    -   arenediyl_((≦12))—;    -   arenediyl_((≦6))—CH₂-arenediyl_((≦6)—; or)

substituted versions of any these groups.

In some embodiments, X is further defined as:

In some embodiments, the third repeat unit is a polyurethane repeatunit. In some embodiments, the third repeat unit is an ether-basedpolyurethane repeat unit. In some embodiments, the third repeat unit isan ester-based polyurethane repeat unit. In some embodiments, thepolymer is an open-celled foam.

In another aspect, the invention provides a wound insert comprising anaptamer-modified polymer or co-polymer as described above or below. Inanother aspect, the invention provides a wound dressing comprising thewound insert and a drape configured to be coupled to skin adjacent awound of a patient. In some embodiments, the wound dressing furthercomprises a fluid delivery pad configured to be coupled to the drape anda fluid source such that the fluid source is actuatable to deliver afluid to a wound through the wound dressing.

In another aspect, the invention provides a wound-treatment apparatuscomprising the wound dressing and a fluid source configured to becoupled to the wound dressing such that the fluid source is actuatableto deliver a fluid to the wound dressing. In some embodiments, theapparatus further comprising a vacuum source configured to be coupled tothe wound dressing such that the vacuum source is actuatable to applynegative pressure to the wound dressing. In some embodiments, the fluidcomprises a functional solution that enhances target binding orfacilitates target release into the wound bed. In some embodiments, thefluid comprises saline solutions, solutions with slightly acidic pH,slightly basic pH, solutions with various surfactants (e.g.,polysorbate), EDTA and/or EGTA. In some embodiments, the fluid compriseshypochlorous acid and hypochlorite ion.

A. DEFINITIONS

As used herein, the term “aptamer” refers not only to oligonucleic acidor peptide molecules that bind to a specific target molecule, but alsoto any ligand that binds to a target molecule or ion. For example, asused herein, “aptamer” would include EDTA (ethylene-diaminetetraaceticacid), which binds to Ca²⁺.

When used in the context of a chemical group, “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “halo” means independently —F, —Cl,—Br or —I; “amino” means —NH₂ (see below for definitions of groupscontaining the term amino, e.g., alkylamino); “hydroxyamino” means—NHOH; “nitro” means —NO₂; imino means=NH (see below for definitions ofgroups containing the term imino, e.g., alkylimino); “cyano” means —CN;“azido” means —N₃; in a monovalent context “phosphate” means —OP(O)(OH)₂or a deprotonated form thereof; in a divalent context “phosphate” means—OP(O)(OH)O— or a deprotonated form thereof; “mercapto” means —SH;“thio” means=S; “thioether” means —S—; “sulfonamido” means —NHS(O)₂—(seebelow for definitions of groups containing the term sulfonamido, e.g.,alkylsulfonamido); “sulfonyl” means —S(O)₂—(see below for definitions ofgroups containing the term sulfonyl, e.g., alkylsulfonyl); and“sulfinyl” means —S(O)—(see below for definitions of groups containingthe term sulfinyl, e.g., alkylsulfinyl).

The symbol “—” means a single bond, “=” means a double bond, and “≡”means triple bond. The symbol “---” represents an optional bond, whichif present is either single or double. The symbol “

” represents a single bond or a double bond. Thus, for example, thestructure

includes the structures

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond. The symbol “

”, when drawn perpendicularly across a bond indicates a point ofattachment of the group. It is noted that the point of attachment istypically only identified in this manner for larger groups in order toassist the reader in rapidly and unambiguously identifying a point ofattachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the conformation (e.g., either R or S) orthe geometry is undefined (e.g., either E or Z).

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the groups and classes below, the following parenthetical subscriptsfurther define the group/class as follows: “(Cn)” defines the exactnumber (n) of carbon atoms in the group/class. “(C≦n)” defines themaximum number (n) of carbon atoms that can be in the group/class, withthe minimum number as small as possible for the group in question, e.g.,it is understood that the minimum number of carbon atoms in the group“alkenyl_((C≦8))” or the class “alkene_((C≦8))” is two. For example,“alkoxy_((C≦10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms). (Cn-n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms)).

The term “saturated” as used herein means the compound or group somodified has no carbon-carbon double and no carbon-carbon triple bonds,except as noted below. The term does not preclude carbon-heteroatommultiple bonds, for example a carbon oxygen double bond or a carbonnitrogen double bond. Moreover, it does not preclude a carbon-carbondouble bond that may occur as part of keto-enol tautomerism orimine/enamine tautomerism.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound/group so modified is an acyclic or cyclic,but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by single bonds(alkanes/alkyl), or unsaturated, with one or more double bonds(alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).When the term “aliphatic” is used without the “substituted” modifieronly carbon and hydrogen atoms are present. When the term is used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or—OC(O)CH₃.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched, cyclo, cyclic or acyclic structure,and no atoms other than carbon and hydrogen. Thus, as used hereincycloalkyl is a subset of alkyl. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂(iso-Pr), —CH(CH₂)₂(cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl),—CH₂CH(CH₃)₂(iso-butyl), —C(CH₃)₃(tert-butyl), —CH₂C(CH₃)₃(neo-pentyl),cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “alkanediyl” when usedwithout the “substituted” modifier refers to a divalent saturatedaliphatic group, with one or two saturated carbon atom(s) as thepoint(s) of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “alkylidene”when used without the “substituted” modifier refers to the divalentgroup ═CRR′ in which R and R′ are independently hydrogen, alkyl, or Rand R′ are taken together to represent an alkanediyl having at least twocarbon atoms. Non-limiting examples of alkylidene groups include: ═CH₂,═CH(CH₂CH₃), and ═C(CH₃)₂. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or —OC(O)CH₃. Thefollowing groups are non-limiting examples of substituted alkyl groups:—CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH, —CH₂C(O)OCH₃, —CH₂C(O)NH₂,—CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂N(CH₃)₂, and—CH₂CH₂Cl. The term “fluoroalkyl” is a subset of substituted alkyl, inwhich one or more hydrogen has been substituted with a fluoro group andno other atoms aside from carbon, hydrogen and fluorine are present. Thegroups, —CH₂F, —CF₃, and —CH₂CF₃ are non-limiting examples offluoroalkyl groups. An “alkane” refers to the compound H—R, wherein R isalkyl.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and no atoms other than carbon and hydrogen.Non-limiting examples of alkenyl groups include: —CH═CH₂ (vinyl),—CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and—CH═CH—C₆H₅. The term “alkenediyl” when used without the “substituted”modifier refers to a divalent unsaturated aliphatic group, with twocarbon atoms as points of attachment, a linear or branched, cyclo,cyclic or acyclic structure, at least one nonaromatic carbon-carbondouble bond, no carbon-carbon triple bonds, and no atoms other thancarbon and hydrogen. The groups, —CH═CH—, —CH═C(CH₃)CH₂—, —CH═CHCH₂—,and

are non-limiting examples of alkenediyl groups. When these terms areused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or—OC(O)CH₃. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, are non-limitingexamples of substituted alkenyl groups. An “alkene” refers to thecompound H—R, wherein R is alkenyl.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl group (carbon number limitation permitting) attached tothe first aromatic ring or any additional aromatic ring present.Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl,(dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, and themonovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic group,with two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl group (carbon number limitation permitting) attached to the firstaromatic ring or any additional aromatic ring present. If more than onering is present, the rings may be fused or unfused. Non-limitingexamples of arenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂ or —OC(O)CH₃. An “arene” refers to the compound H—R,wherein R is aryl.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn) and 2-phenyl-ethyl. When the term is used with the“substituted” modifier one or more hydrogen atom from the alkanediyland/or the aryl has been independently replaced by —OH, —F, —Cl, —Br,—I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂ or —OC(O)CH₃. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of an aromatic ring structure wherein at least one ofthe ring atoms is nitrogen, oxygen or sulfur, and wherein the groupconsists of no atoms other than carbon, hydrogen, aromatic nitrogen,aromatic oxygen and aromatic sulfur. As used herein, the term does notpreclude the presence of one or more alkyl group (carbon numberlimitation permitting) attached to the aromatic ring or any additionalaromatic ring present. Non-limiting examples of heteroaryl groupsinclude furanyl, imidazolyl, indolyl, indazolyl (Im), methylpyridyl,oxazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl,quinoxalinyl, thienyl, and triazinyl. The term “heteroarenediyl” whenused without the “substituted” modifier refers to an divalent aromaticgroup, with two aromatic carbon atoms, two aromatic nitrogen atoms, orone aromatic carbon atom and one aromatic nitrogen atom as the twopoints of attachment, said atoms forming part of one or more aromaticring structure(s) wherein at least one of the ring atoms is nitrogen,oxygen or sulfur, and wherein the divalent group consists of no atomsother than carbon, hydrogen, aromatic nitrogen, aromatic oxygen andaromatic sulfur. As used herein, the term does not preclude the presenceof one or more alkyl group (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. If more than one ring is present, the rings may be fused orunfused. Non-limiting examples of heteroarenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂ or —OC(O)CH₃.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, aryl, aralkyl orheteroaryl, as those terms are defined above. The groups, —CHO, —C(O)CH₃(acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃, —C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂,—C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅, —C(O)(imidazolyl) arenon-limiting examples of acyl groups. A “thioacyl” is defined in ananalogous manner, except that the oxygen atom of the group —C(O)R hasbeen replaced with a sulfur atom, —C(S)R. When either of these terms areused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or—OC(O)CH₃. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃(methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and —CON(CH₃)₂, arenon-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃, —OCH₂CH₃,—OCH₂CH₂CH₃, —OCH(CH₃)₂, —OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl.The terms “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”,“heteroaryloxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as —OR, in which R is alkenyl,alkynyl, aryl, aralkyl, heteroaryl, and acyl, respectively. Similarly,the term “alkylthio” when used without the “substituted” modifier refersto the group —SR, in which R is an alkyl, as that term is defined above.The term “alkoxydiyl” when used without the “substituted” modifierrefers to the divalent group —O-alkanediyl-. When any of these terms isused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or—OC(O)CH₃. The term “alcohol” corresponds to an alkane, as definedabove, wherein at least one of the hydrogen atoms has been replaced witha hydroxy group.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃ and —NHCH₂CH₃. The term “dialkylamino” when used without the“substituted” modifier refers to the group —NRR′, in which R and R′ canbe the same or different alkyl groups, or R and R′ can be taken togetherto represent an alkanediyl. Non-limiting examples of dialkylamino groupsinclude: —N(CH₃)₂, —N(CH₃)(CH₂CH₃), and N-pyrrolidinyl. The terms“alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, and “alkylsulfonylamino” when usedwithout the “substituted” modifier, refers to groups, defined as —NHR,in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, andalkylsulfonyl, respectively. A non-limiting example of an arylaminogroup is —NHC₆H₅. The term “amido” (acylamino), when used without the“substituted” modifier, refers to the group —NHR, in which R is acyl, asthat term is defined above. A non-limiting example of an amido group is—NHC(O)CH₃. The term “alkylimino” when used without the “substituted”modifier refers to the divalent group ═NR, in which R is an alkyl, asthat term is defined above. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or —OC(O)CH₃. The groups—NHC(O)OCH₃ and —NHC(O)NHCH₃ are non-limiting examples of substitutedamido groups.

The terms “alkylsulfonyl” and “alkylsulfinyl” when used without the“substituted” modifier refers to the groups —S(O)₂R and —S(O)R,respectively, in which R is an alkyl, as that term is defined above. Theterms “alkenylsulfonyl”, “alkynylsulfonyl”, “arylsulfonyl”,“aralkylsulfonyl”, and “heteroarylsulfonyl”, are defined in an analogousmanner. When any of these terms is used with the “substituted” modifierone or more hydrogen atom has been independently replaced by —OH, —F,—Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or —OC(O)CH₃.

The term “glycoside” refers to a compound in which a sugar group isbound to a non-carbohydrate moiety. Typically the sugar group (glycone)is bonded through its anomeric carbon to another group (aglycone) via aglycosidic bond that has an oxygen, nitrogen or sulfur atom as a linker.

A “simple sugar” are the basic structural units of carbohydrates, whichcannot be readily hydrolyzed into simpler units. The elementary formulaof a simple monosaccharide is C_(n)H_(2n)O_(n), where the integer n isat least 3 and rarely greater than 7. simple monosachharides may benamed generically according on the number of carbon atoms n: trioses,tetroses, pentoses, hexoses, etc. Simple sugars may be open chain(acyclic), cyclic or mixtures thereof. In these cyclic forms, the ringusually has 5 or 6 atoms. These forms are called furanoses andpyranoses, respectively—by analogy with furan and pyran. Simple sugarsmay be further classified into aldoses, those with a carbonyl group atthe end of the chain in the acyclic form, and ketoses, those in whichthe carbonyl group is not at the end of the chain. Non-limiting examplesof aldoses include: glycolaldehyde, glyceraldehydes, erythrose, threose,ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose,gulose, idose, galactose and talose. Non-limiting examples of aldosesinclude: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose,psicose, sorbose and tagatose. The ‘D-’ and ‘L-’ prefixes may be used todistinguish two particular stereoisomers which are mirror-images of eachother. The term simple sugar also covers O-acetyl derivatives thereof.

An “amino sugar” or “aminoglycoside” refers to a derivative of a sugar,deoxy sugar, sugar acid or sugar alcohol, where one or more hydroxygroup(s) has been replace with one more amino group(s). A “simple aminosugar” refers to a derivative of a simple sugar, simply deoxy sugar,simply sugar acid or sugar alcohol, where one or more hydroxy group(s)has been replace with one more amino group(s). These terms also cover N-and O-acetyl derivatives thereof. Non-limiting examples includeN-acetylglucosamine, galactosamine, glucosamine and sialic acid.

The term “deoxy sugar” refers to a sugar derivative where one of thehydroxy groups of a carbohydrate has been replaced with a hydrogen atom.A “simple deoxy sugar” is a deoxy sugar derived from a simple sugar, asdefined herein. These terms also cover O-acetyl derivatives thereof.Non-limiting examples of simple deoxy sugars are deoxyribose (based uponribose), fucose, and rhamnose.

The term “sugar acid” refers to a sugar derivative where an aldehydefunctional group or one or more hydroxy functional groups has beenoxidized to a carboxyl group. Aldonic acids are those in which thealdehyde functional group of an aldose has been oxidized. Ulosonic acidsare those in which the first hydroxyl group of a 2-ketose has beenoxidized creating an t-ketoacid. Uronic acids are those in which theterminal hydroxyl group of an aldose or ketose has been oxidized.Aldaric acids are those in which both ends of an aldose have beenoxidized. Non-limiting aldonic acids include glyceric acid (3C), xylonicacid (5C), gluconic acid (6C), and ascorbic acid (6C, unsaturatedlactone). Non-limiting examples of ulosonic acids include neuraminicacid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid) andketodeoxyoctulosonic acid (KDO or 3-deoxy-D-manno-oct-2-ulosonic acid).Non-limiting examples of uronic acids include glucuronic acid (6C),galacturonic acid (6C), and iduronic acid (6C). Non-limiting example ofaldaric acids include tartaric acid (4C), meso-galactaric acid (mucicacid) (6C), and D-glucaric acid (saccharic acid) (6C). A “simple sugaracid” is a sugar acid derived from a simple sugar. These terms alsocover O-acetyl derivatives thereof.

The term “sugar alcohol” refers to a sugar derivative whose carbonylgroup (aldehyde or ketone, reducing sugar) has been reduced to a primaryor secondary hydroxyl group. Non-limiting examples of sugar alcoholsinclude: glycol (2-carbon), glycerol (3-carbon), erythritol (4-carbon),threitol (4-carbon), arabitol (5-carbon), xylitol (5-carbon), ribitol(5-carbon), mannitol (6-carbon), sorbitol (6-carbon), dulcitol(6-carbon), iditol (6-carbon), isomalt (12-carbon), maltitol(12-carbon), lactitol (12-carbon) or polyglycitol. A “simple sugaralcohol” is a sugar alcohol derived from a simple sugar. These termsalso cover O-acetyl derivatives thereof.

As used herein, the term “monosaccharide group” refers to a monovalentcarbohydrate group, with a carbon atom as the point of attachment. Theterm covers the groups resulting from removal of a hydroxyl radical froma simple sugar (e.g., glucose), simple deoxy sugar (e.g., fucose),simple sugar acid (e.g., gluconic acid), simple sugar alcohol (e.g.,xylitol) or simple amino sugar (e.g., glucosamine). Typically themonosaccharide group is bonded through its anomeric carbon to anothergroup (aglycone) via oxygen atom linker. In some cases the linker may bea nitrogen or sulfur atom.

A “disaccharide group” is a monovalent carbohydrate group consisting oftwo monosaccharide groups, wherein the second monosaccharide groupreplaces a hydrogen on a hydroxy group of the first monosaccharidegroup. Non-limiting examples of disaccharide groups include thosederived from sucrose, lactulose, lactose, maltose trehalose andcellobiose.

A “trisaccharide group” is a monovalent carbohydrate group consisting ofthree monosaccharide groups, wherein the second monosaccharide groupreplaces a hydrogen on a hydroxy group of the first monosaccharide groupand the third monosaccharide group replaces a hydrogen on a hydroxygroup of either the first or the second monosaccharide groups.

An oligosaccharide is a monovalent carbohydrate group consisting ofthree to ten, preferably three to six monosaccharide groups, wherein thesecond monosaccharide replaces a hydrogen on a hydroxy group of thefirst monosaccharide, the third monosaccharide replaces a hydrogen on ahydroxy group of either the first or the second monosaccharide groups,and subsequent monosaccharide groups replace hydrogens on any previouslyjoined monosaccharide groups, thus forming either a linear or branchedstructure.

The term “silyl” when used without the “substituted” modifier refers tothe group —SiR₃, where each R is independently hydrogen or unsubstitutedalkyl, as that group is defined above. The term “substituted silyl”refers to the group, —SiR₃, wherein at least one of the R groups and asmany as all of the R groups, is independently a substituted alkyl or—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂ or —OC(O)CH₃. Any remaining Rgroups of the substituted silyl group are independently hydrogen orunsubstituted alkyl. The term “silylated” or “silanated” indicates thata given compound has been derivatized to contain a silyl and/orsubstituted silyl group. The abbreviation “—Sil” refers to silyl and/orsubstituted silyl groups.

The term “coupled” is defined as connected, although not necessarilydirectly, and not necessarily mechanically; two items that are “coupled”may be integral with each other. The terms “a” and “an” are defined asone or more unless this disclosure explicitly requires otherwise. Theterms “substantially,” “approximately,” and “about” are defined aslargely but not necessarily wholly what is specified, as understood by aperson of ordinary skill in the art.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “include” (and any form of include, such as “includes” and“including”) and “contain” (and any form of contain, such as “contains”and “containing”) are open-ended linking verbs. As a result, a methodthat “comprises,” “has,” “includes” or “contains” one or more stepspossesses those one or more steps, but is not limited to possessing onlythose one or more steps. Likewise, a wound dressing that “comprises,”“has,” “includes” or “contains” one or more elements possesses those oneor more elements, but is not limited to possessing only those elements.For example, in a wound dressing that comprises a wound insert and adrape, the wound dressing includes the specified elements but is notlimited to having only those elements. For example, such a wounddressing could also include a connection pad configured to be coupled toa wound-treatment apparatus.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

“PBS” is phosphate buffered saline; “phr” is parts per hundred resin;“OPA” is ortho-pthaldialdehyde; “RT” is room temperature.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

As used herein, the term “polymer” includes “copolymers.”

A “repeat unit” is the simplest structural entity of certain materials,for example, frameworks and/or polymers, whether organic, inorganic ormetal-organic. In the case of a polymer chain, repeat units are linkedtogether successively along the chain, like the beads of a necklace. Forexample, in polyethylene, —[CH₂CH₂]_(n)—, the repeat unit is —CH₂CH₂—.The subscript “n” denotes the degree of polymerization, that is, thenumber of repeat units linked together. When the value for “n” is leftundefined or where “n” is absent, it simply designates repetition of theformula within the brackets as well as the polymeric nature of thematerial. The concept of a repeat unit applies equally to where theconnectivity between the repeat units extends three dimensionally, suchas in metal organic frameworks, modified polymers, thermosettingpolymers, etc.

The term “reticulated open cell foam” or ROCF refers to a foam materialwith a porous structure consisting of an interconnected network of solidstruts. The open cells are formed by the reticulation process, which isin turn defined as in the form of a network or having a network ofparts. Because all struts are connected, the open cell porosity is alsoconnected creating a continuously porous material. The ROCF can bedefined specifically by three independent properties; pore size,relative density, and base material. In some embodiments, ROCF is madefrom polyurethane.

GM-CSF Granulocyte Macrophage Colony Stimulating Factor P22 Commerciallyavailable anti-GM-CSF Peptide ROCF Reticulated open cell foam VEGFVascular endothelial growth factor P16 Anti-VEGF peptide designed denovo 602 Gelest Compound SIA0602.0, a monoethoxysilane 630 GelestCompound SIA0630.0, a triethoxysilane RFUs Relative Fluorescent UnitsTRITC Tetramethylrhodamine isothiocyanate, commonly used for fluorescentmicroscopy Sulfo-EMCS ([N-e-Maleimidocaproyloxy] sulfosuccinimide ester)NHS ester N-hydroxysuccinimide ester EtOH Ethanol Ex/EmExcitation/Emission OPA Ortho-pthaldialdehyde DI H₂O Deionized water RTRoom Temperature aa Amino acid ELISA Enzyme-linked immunosorbent assayEtOH Ethanol

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

Further, a device or structure that is configured in a certain way isconfigured in at least that way, but it can also be configured in otherways than those specifically described.

B. APTAMER MODIFIED MATERIALS

In one aspect the present invention provides a covalent system forcatching and concentrating specific factors in the wound environment,including factors that trigger a significant biological response, suchas proliferation, differentiation, or angiogenesis. This technology maybe used, for example, to deliver biomolecules or bioactive compounds tothe wound bed. Aptamer modified materials provided herein may be used insome embodiments as a system for catching and concentrating specificfactors in the wound environment, for example, factors triggering abiological response such as angiogenesis, or to deliver endogenous orexogenous biomolecules or bioactive compounds to the wound bed.

In one aspect, the aptamer modified materials are provided comprising atleast three components: (1) a polymer, (2) at least one linkercovalently attached to the polymer, e.g., directly, through a side chainof the polymer, or through another linker molecule connected to thepolymer, and (3) at least one aptamer molecule, which may be covalentlyattached to the polymer backbone, but will be more typically covalentlyattached to a linker.

-   -   1) Suitable Polymers

Polymers for use with the present invention include hydrophobic orhydrophilic polyurethanes, chitosan, crosslinked and/or uncrosslinkedpolyolefins, polyols, ethylene vinyl acetate (EVA), elastomers such asacrylonitrile butadiene (NBR), polychloroprene (PCP or CR), ethylenepropylene rubber (EPR & EPDM), poloxamers, silicones, and/or fluorocarbon polymers. For example, in some embodiments, a chitosan-basedpolyurethane polymer may be used.

Polyurethanes are reaction polymers. A urethane linkage is produced byreacting an isocyanate group, —N═C═O with a hydroxy group, andpolyurethanes are produced by the polyaddition reaction of apolyisocyanate with a diol or a polyol, typically in the presence of acatalyst and other additives. A polyisocyanate is a molecule with two ormore isocyanate functional groups, R—(N═C═O)_(n), wherein n≧2 and apolyol is a molecule with two or more hydroxyl functional groups,R′—(OH)_(n), wherein ≧2. The reaction product is a polymer containingthe urethane linkage, —RNHCOOR′—. Polyurethanes may be produced byreacting a liquid isocyanate with a liquid blend of polyols, catalyst,and other additives. The blend of polyols and other additives may alsobe called a resin or a resin blend. In some embodiments, resin blendadditives may include chain extenders, cross linkers, surfactants, flameretardants, blowing agents, pigments, and/or fillers. The synthesis ofbreathable or air-permeable, open cell, flexible urethane polymers istaught for example by U.S. Pat. No. 5,686,501, which is incorporated byreference herein in its entirety.

Molecules that contain two isocyanate groups are called diisocyanates.Isocyanates may be classed as aromatic, such as diphenylmethanediisocyanate (MDI), diphenylethane diisocyanate (EDI), or toluenediisocyanate (TDI); or aliphatic, such as hexamethylene diisocyanate(HDI) or isophorone diisocyanate (IPDI). An example of a polymericisocyanate is polymeric diphenylmethane diisocyanate, which is a blendof molecules with two-, three-, and four- or more isocyanate groups.Isocyanates can be further modified by partially reacting them with apolyol to form a prepolymer. A “quasi-prepolymer” is formed when thestoichiometric ratio of isocyanate to hydroxyl groups is greater than2:1. A “true prepolymer” is formed when the stoichiometric ratio isequal to 2:1. Important characteristics of isocyanates include theirmolecular backbone, % NCO content, functionality, and viscosity.

Molecules that contain two hydroxyl groups are called diols. Examplesinclude, ethylene glycol (EG), 1,4-butanediol (BDO), diethylene glycol(DEG). Molecules that contain three hydroxyl groups are called triols.Examples include glycerol. Polyols may themselves be polymers. Forexample, they may be formed by base-catalyzed addition of propyleneoxide (PO), ethylene oxide (EO) onto a hydroxy or amino-containinginitiator, or by polyesterification of a di-acid, such as adipic acid,with glycols, such as ethylene glycol or dipropylene glycol (DPG).Polyols extended with PO or EO are typically called polyether polyols.Polyols formed by polyesterification are typically called polyesterpolyols. The choice of initiator, extender, and molecular weight of thepolyol will typically affect its physical state, and the physicalproperties of the resulting polyurethane. Important characteristics ofpolyols are their molecular backbone, initiator, molecular weight, %primary hydroxyl groups, functionality, and viscosity.

One attribute of polyurethanes is its ability to be turned into foam.Small amounts of water may also be added to accomplish this in someembodiments. Water will react with isocyanate to create carbon dioxidegas, which fills and expands cells created during the mixing process.Blowing agents may also be used, including certain halocarbons such asHFC-245fa (1,1,1,3,3-pentafluoropropane) and HFC-134a(1,1,1,2-tetrafluoroethane), and hydrocarbons such as n-pentane. In someembodiments, surfactants may be used to modify the characteristics ofthe polymer during the foaming process.

Though the properties of the polyurethane are typically determinedmainly by the choice of polyol, the diisocyanate exerts some influence.For example, the cure rate will generally be influenced by thereactivity of a given functional group and the number of functionalisocyanate groups.

Softer, elastic, and more flexible polyurethanes typically result whenlinear difunctional polyethylene glycol segments, commonly calledpolyether polyols, are used to create the urethane links. More rigidproducts typically result if polyfunctional polyols are used, as thesecreate a three-dimensional cross-linked structure which, again, can bein the form of a low-density foam. Control of viscoelastic properties,for example, by modifying the catalysts and polyols used can lead tomemory foam, which is much softer at skin temperature than at roomtemperature.

In some embodiments of the present invention, the polyurethane foam isformed by the polymerization of isocyanates and polyols, typicallytoluene diisocyanate and a multi arm polyether polyol. These componentsmay be indexed, such that the isocyanate and hydroxyl group are in a oneto one ratio.

Chitosan is a linear polysaccharide derived from the exoskeletons ofcrustaceans, which can be represented in some embodiments by thefollowing formula:

In some embodiments, chitosan is a linear polysaccharide composed ofrandomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit)and N-acetyl-D-glucosamine (acetylated unit). The presence of thehydroxy groups and amino groups allows the utilization of a wide varietyof conjugation chemistries. For example, chitosan may be polymerizedand/or co-polymerized with diisocyanate or triisocyanate molecules,including diisocyanate or trisiocyanate prepolymers, into a polyurethanepolymer or co-polymer.

In some embodiments a polyol other than chitosan may be used in additionto the chitosan. Addition of chitosan to a polyol resin creates urethanebonds between the isocyanate and the hydroxyl groups on the chitosanring. The resulting foam contains chitosan segments to be available onthe struts of the foam and gives the foam some of the properties ofchitosan. In addition the addition of chitosan produces an over-indexedfoam, with surplus hydroxyl groups and amine groups on the chitosansegments that are available for additional surface chemistry. Forexample, FIG. 7 depicts a repeat units of polyurethane based on thepolymerization of TDI, chitosan, and Voranol™ 3010 triol. In someembodiments chitosan or another diol or polyol may be polymerized intopolyurethane at a ratio of 0.05-0.5 g chitosan per 1.0 g of adiisocyanate molecule or prepolymer. Further details and examples ofsuch procedures are provided in the Examples section below.

Formulations of 5, 10, 20 and 30 PHR (grams of chitosan Per Hundredgrams of polyurethane Resin) have been produced and tested. They havebeen characterized by the amount of surface amine available for bondingby conjugation of the foam with o-phthaldialdehyde. The relativefluorescence units RFU were found to be proportional to the primaryamine available on the surface of the foam.

-   -   2) Linker-Modified Polymer Backbone

In some embodiments, the invention provides substituted silyl-modifiedpolymers. Examples include those comprising repeat units based onpolyurethane, which may be hydrophobic or hydrophilic, chitosan,crosslinked and/or uncrosslinked polyolefin, polyols, ethylene vinylacetate (EVA), elastomers such as acrylonitrile butadiene (NBR),polychloroprene (PCP or CR), ethylene propylene rubber (EPR & EPDM),silicones, and/or fluoro carbon polymers. For example, in someembodiments, a chitosan-based polyurethane polymers or copolymers may beused. In some embodiments, the substituted silyl groups are attached tothe polymer or co-polymer through an oxygen atom. In other embodiments,the substituted silyl groups are attached directly to a carbon atom ofthe polymer or co-polymer.

In some embodiments, the present invention provides polymeric materialsmodified with substituted silyl groups, for example,(H₂N(CH₂)₃)(i-Pr)₂Si— and (H₂N(CH₂)₁₁)(EtO)₂Si—. In some embodiments,the materials of the present invention comprise a polymeric foam.Examples of foam materials can include polyurethane-based foam, whichmay be hydrophobic or hydrophilic, including, for example achitosan-based polyurethane foam material. Other foams that may besuitable include chitosan, crosslinked and/or uncrosslinkedpolyolefin's, polyols, ethylene vinyl acetate (EVA), and elastomers suchas acrylonitrile butadiene (NBR), polychloroprene (PCP or CR), ethylenepropylene rubber (EPR & EPDM), silicones, and fluoro carbon polymers.For example, in some embodiments, a chitosan-based polyurethane foam maybe used. In some embodiments, the substituted silyl groups are attachedto the foam through an oxygen atom. In other embodiments, thesubstituted silyl groups are attached directly to a carbon atom of thefoam.

The silylated polyurethane foams disclosed herein may be made bysilylating the hydroxy groups on a polymer comprising such groups. Asused herein, silylation is the introduction of a substituted silyl group(R₃Si—) to a molecule. It involves the replacement of a hydrogen on thecompound, e.g., the hydrogen of a hydroxy group, with an substitutedsilyl group, for example, the compound3-aminopropyldiisopropylethoxysilane will react with hydroxy groupsunder the appropriate conditions such groups to form a new covalent Si—Obond thereby linking the H₂N(CH₂)₃Si(iPr)₂— group to an oxygen which isin turn attached to the point of attachment on the molecule where thehydroxy group had been previously attached. Without being bound bemechanism, the oxygen atom of the product, may be the same oxygen atomof the hydroxy group reactant. See “How do I apply my Silane?” GelestCatalog. 2006, pages 19-20, which are incorporated by reference hereinin their entirety.

Example 1 below describes in detail how amino-substituted silyl groupsmay be deposited on 30 PHR chitosan foam. Example 2, describes howmaleimide-thiol conjugation can be performed with Sulfo-EMCS, which hasthe formula:

In some embodiments, the N-hydroxysuccinimide ester (NHS ester) on oneend of the Sulfo-EMCS molecule can react with free amino groups of thelinker or polymer. The maleimide group on the other end of the moleculemay be used to react, for example, with —SH groups on a peptide aptamerto form stable thioether bonds. In this manner the Sulfo-EMCS may beused to link the peptide to a polymer or copolymer, including a foamsubstrate.

Furthermore, the peptides used in maleimide-thiol conjugation typicallyhave specially modified C-termini. For example, the C-termini may be“capped” with Cys residues bearing reactive —SH groups to facilitate theconjugation. In a Sulfo-EMCS conjugations, Sulfo-EMCS is typically usedin excess, for example, in 50-100× molar excess relative to anorganosilane crosslinkers to facilitate the reaction. Such reactions maybe carried out, for example, at pH 7.0-7.5 and at room temperature. Suchmethods may be further modified and optimized using the principles andtechniques of organic chemistry as applied by a person skilled in theart. Such principles and techniques are taught, for example, in March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007),which is also incorporated by reference herein in its entirety. Forexample, other suitable amino-thio linkers may be used. These include,for example: SM(PEG)24, SM(PEG)12, SM(PEG)8, SM(PEG)6, SM(PEG)4,Sulfo-LC-SMPT, SM(PEG)2, Sulfo-KMUS, LC-SMCC, LC-SPDP, Sulfo-LC-SPDP,SMPH, Sulfo-SMPB, SMPB, SMPT, SIAB, Sulfo-SIAB, EMCS, Sulfo-SMCC, SMCC,MBS, GMBS, Sulfo-GMBS, Sulfo-MBS, SPDP, SBAP, BMPS, AMAS, and SIA.

In some embodiments of the present invention, the silylated polymer willbe further modified with additional linker molecules, for example,oligopeptide oligomers. In some embodiments, the additional linkers arecovalently attached to the backbone of the silylated polymer orcopolymer. In other embodiments, the linker is covalently attached to aside chain or side group of the polymer or copolymer. In someembodiments, the additional linker is attached via a function group(e.g., an amino group) of a substituted silyl group of the silylatedpolymer or copolymer, forming a material-linker₁-linker₂ modifiedmaterial, wherein the linke₁ is a substituted silyl group (a.k.a.heterobifunctional silane crosslinker) such as those discussed herein,and linker₂ may be another a second substituted silyl group or anothertype of linker molecule, such an oligopeptide. In some embodiments,linker₂ may formed from the reaction of a terminal amino or a mercaptogroup on linke₁ with EMCS (N—[ε-maleimidocaproyloxy]succinimide ester).See Example 2 below.

Further functionalization of the interfacial layer created by, forexample, silylation can be achieved by linking the other end of thesilyl group or linker₂ group to aptamers, including “capture peptides”as discussed further below.

-   -   3) Aptamer-Linker-Modified Polymer Backbone

In some embodiments, aptamers, including peptide-based aptamers, may beconjugated to a polymer or copolymer. Peptide aptamers also referred toas “capture peptides” herein, can be used to bind protein targets in thewound environment without inhibiting their signaling capabilities orother functions. Such capture peptides may also be used to capture andincrease concentrations of biologically active proteins across a woundbed of a patient, for example, to strengthen or activate a targetedbiological response. These and other uses are described in greaterdetail below.

In some embodiments, a linker is further connected to one or moredifferent types of aptamers, forming a polymer backbone-linker-aptamerconjugate. In some embodiments, the linker is covalently attached to thebackbone of the polymer. In other embodiments, the linker is covalentlyattached to a side group of the polymer. In some embodiments, theaptamer is directly linked to the backbone of the polymer, forming apolymer backbone-aptamer conjugate.

Poloxamer-EMCS-peptide constructs were also successfully used to bindaptamers to a foam. The hydrophobic portion of the poloxamer has stronghydrophobic interactions with the foam. The hydrophilic “arms” has aminogroups, which were then linked to oligopeptides using and EMCS linker.

In some embodiments, the aptamer is a computationally selected or phagedisplay derived “capture peptide.” Such “capture peptides” may be usedto target specific proteins or protein families for binding. One exampleof such a peptide is P-16, defined by the amino acid sequenceGHQGQHIGQMS-AEEAc-Cys-NH₂, where the AEEAc-Cys-NH₂ is a speciallymodified C-terminus for conjugation to maleimide groups provided by theEMCS linker. P-16 has been shown to bind to VEGF and PDGF, andpotentially other members of the PDGF super-family.

Example 2 below reports the conjugation of an aptamer to 30 PHR chitosanfoam via a Sulfo-EMCS ([N-ε-maleimidocaproyloxy] sulfosuccinimide ester)crosslinkers.

For example, once bound, the terminal amino group of the3-aminopropyldiisopropylethoxysilane remains accessible for capturepeptide or aptamer binding. In some embodiments biologically activepeptides may be bound to this terminal amine group in a manner as totheir retain their biological activity. These attached peptides, whenthe material is used, for example, as part of a dressing, a wound insertor pad, may be used to capture and increase concentrations ofbiologically active proteins across a wound bed strengthening oractivating the targeted biological response. For example, dressings maybe prepared to concentrate proteins such as vascular endothelial growthfactor in the wound bed, to trigger angiogenesis, and/or to triggertubule formation. In this manner such materials may be used to bettercontrol various biological pathways in a wound environment

When a peptide or a factor is attached to the other end of the silanecross linker, it will have a dissociation constant (K_(d)), or bindingaffinity, that is specific to a given target molecule at a given set ofconditions. In some embodiments, it may allow for reversible binding ofone or more target(s). In some embodiments, the aforementioned targetmay be released back into a wound when used as part of the methods anddevices contemplated herein. In some embodiments, ingredients may beadded to the instillation solution to not only help dissociate the boundfactor back into the wound environment but to also interactsynergistically with the retained exudate element(s) for a modulatingeffect so that a favorable wound healing response is elicited. Types ofinstillation solutions are discussed in greater detail below.

Examples of instilled ingredients which may be used in some embodimentsto dissociate bound molecules from the peptide linkers include: salinesolutions, solutions with slightly acidic pH, slightly basic pH,solutions with various surfactants (i.e. polysorbate), EDTA or EGTA. Insome embodiments, the fluid instilled to initiate the dissociation ofthe bound factors from the linker will depend upon the binding strengthof the factor-linker complex, which is in turn determined by thedissociation constant. The dissociation constant may be modified byusing knowledge of amino acid chemistry of the factor of interest todesign the linker/peptide/aptamer.

One example of a modified dressing or wound insert is one capable ofbinding Ca²⁺, wound derived or otherwise, and retaining it at the woundsite. During the early phases of wound healing, Ca²⁺ ions are typicallyreleased from the cells locally into the extracellular space. Theresulting high Ca²⁺ concentration is believed to be a positive effectorof many cellular processes involved in wound healing such as adhesion,migration and differentiation. When a high Ca²⁺ concentration isrequired or is beneficial to the wound bed, instillation or flushingwith a solution containing a chelator (e.g., EDTA) may be used todisrupt the binding of Ca²⁺ to the dressing and into the wound bed.

Another factor for use with the current invention is transferrin, ablood plasma protein for iron binding. Chronic wound fluid has beenshown to have significantly lower transferrin levels indicating thatoxidative stress occurs in chronic wounds. It is known that free ironcan play a role in the formation of free radicals. Without being boundby theory, high levels of free iron may contribute to exacerbation oftissue damage and delayed wound healing. Binding and concentratingtransferrin onto the dressing can be used to sequester free iron in thewound bed with subsequent release with an appropriate instillationsolution and subsequent removal with the exudate following the use ofnegative pressure. This specific time-dependent modulation oftransferring and iron levels can provide a significant benefit to thepatient. In an alternate view, the affinity of transferrin for iron isvery high but is reversible in that it decreases progressively withdecreasing pH below neutrality. If a need for localized ironconcentration is necessary, instillation of a low pH solution can beused to unbind or release iron from transferrin.

Hyaluronan, or hyaluronic acid, is another possible target contemplatedfor use with the current invention. Without being bound by theory,immobilizing and concentrating hyaluronan to the wound bed beforeinstillation with an appropriate solution for release may be used tocontribute to keratinocyte proliferation and migration and reducecollagen deposition, which in turn is known to lead to reduced scarring.Hyaluronan is also known for its free-radical scavenging function thatcould be beneficial as it is bound to the foam on the wound site.

Lactoferrin, known for its antimicrobial and anti-inflammatoryproperties, is another possible target for some of the embodiments ofthe present invention. Secreted by endothelial cells, lactoferrin hasbeen shown to have a synergistic effect with FGF2 in that there is amarked increase in their ability together to effect fibroblast migrationand proliferation. Specifically designed aptamers can be used to bindboth LF and FGF2 and release them with an appropriate instillationsolution in an opportune therapeutic timeframe.

TGFB-3 can be another target another possible target for some of theembodiments of the present invention. This protein promotesreorganization of matrix molecules, resulting in an improved dermalarchitecture and reduced scarring. TGFB-3 is secreted in latent formthat requires activation before it is functional. Activation of latentTGFB-3 occurs via binding to thrombospondin-1 (TSP-1). Therefore, TSP-1,may be used in some embodiments, as an ingredients in the instillationfluid to modulate TGFB-3 activity.

Possible other targets include calmodulin, S-100, thyroxine and cholatereceptors, amongst many others.

C. USES OF APTAMER MODIFIED MATERIALS

The modified polymers described above and materials made therefrom maybe used for a variety of purposes, including to (a) capture andconcentrate biological targets in the wound environment, (b) specify thechemical nature of the binding, and/or (c) dictate the orientation withwhich the target factors are presented to the cells. As discussed ingreater detail in the Examples section below, aptamer-modifiedpolyurethanes, including peptide modified GranuFoam™ (GF), a type ofROCF, may be used to capture specific protein targets in vitro. Forexample, peptides P16, an anti VEGF peptide, and P22, anti-GM-CSFpeptide, were covalently bound to ROCF, and it was demonstrated thatthese aptamers capture target proteins. Examples 2 and 3 providesresults quantifying the amount of GM-CSF captured by ROCF coated withP22, and Example 4 presents results quantifying the amount of VEGFcaptured by ROCF coated with P16.

In some embodiments the aptamer-modified polymers may be used to bindprotein targets in the wound environment without inhibiting theirsignaling capabilities or other functions. Such aptamer-modifiedpolymers may be used as part of a dressing or wound insert, for example,to capture and increase concentrations of biologically active proteinsacross a wound bed of a patient, for example, to strengthen or activatea targeted biological response.

In some embodiments, such a dressing may be used to concentrate proteinsof interest such as vascular endothelial growth factor in the wound bedto trigger angiogenesis and tubule formation. Additionally, peptides maybe designed to antagonize or sequester proteins that adversely affectthe healing process, such as matrix metalloproteinases. Dressings madeof such aptamer-modified polymers may thus be used, in some embodiments,to modulate various biological pathways or to manage the presence ofunwanted bioactive molecules or enzymes in the wound environment.

In addition to dressings, aptamer-modified polymers, including thoseusing a heterobifunctional silyl-modified linker and apolyurethane-based polymer or copolymer, may also be used in a widearray of other materials, matrixes and biomedical devices, includingcatheters. In such embodiments, they may be used to conjugate a varietyof aptamers, or other compounds, including antimicrobials. Theapplication of these materials to a negative pressure-based therapy isdiscussed in greater detail below.

Materials of this invention may also have the advantage that they may bemore efficacious than, be less toxic than, be longer acting than, bemore potent than, produce fewer side effects than, be more easilyabsorbed than, and/or have a better pharmacokinetic profile (e.g.,higher oral bioavailability and/or lower clearance) than, and/or haveother useful pharmacological, physical, or chemical properties over,compounds known in the prior art, whether for use in the indicationsstated herein or otherwise.

It should be recognized that the particular anion or cation forming apart of any salt of this invention is not critical, so long as the salt,as a whole, is pharmacologically acceptable. Additional examples ofpharmaceutically acceptable salts and their methods of preparation anduse are presented in Handbook of Pharmaceutical Salts: Properties, andUse (2002), which is incorporated herein by reference.

D. PREPARATION OF INSERTS COMPRISING FOAM MATERIALS BASED

In another aspect, foam-based polymers may be physically and/orchemically treated, coated or manipulated before or after they arecovalently linked to an aptamer. Some embodiments of making modifiedwound inserts comprise: compressing (and/or felting) at least a portionof a foam. Some embodiments comprise: treating (e.g., by applying heat,or activating a coating that has been applied to) the compressed foamsuch that the foam remains substantially compressed in the absence of anexternal compressive force. For example, in some embodiments, treatingcomprises heating the foam (e.g., foam) to an elevated temperaturesufficient to reduce resiliency of the foam. For example, the foam canbe heated to a temperature at which resiliency of the foam is reducedand/or relaxed, but that is below the melting temperature of the foam(e.g., such that the foam is not degraded by the elevated temperature).In this way, the foam can be compression set using heat and pressure(compressive force) to relax compressive strains developed in the foam.Generally, high temperatures are used to achieve the compression set. Toachieve the desired “set” such that resiliency of the foam is reducedand/or the foam remains substantially compressed in the absence of acompressive force, temperatures can range from 158 degrees Fahrenheit to482 degrees Fahrenheit (e.g., equal to, less than, greater than, orbetween any of: 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340,360, 380, 400, 420, 440, 460, 480, 500 degrees Fahrenheit, dependingupon the particular foam used). The foam may also be put through acooling cycle to help retain the set introduced. For example, the foammay be cooled to a temperature below room or ambient temperature (e.g.,to or in a temperature equal to, less than, greater than, or between anyof: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 degreesFahrenheit). In some embodiments of the present methods of forming amodified wound insert, the foam is disposed between two heated plates orplatens (e.g., in a plate or platen press and/or where the plates areheated to a temperature sufficient to reduce the resiliency of thefoam); and the press is actuated to move the plates toward one another(e.g., perpendicular to thickness 320 of thick portions 304) such thatthe foam is compressed to the desired overall thickness or degree ofcompression). Such a press can be electrically, mechanically, and/orhydraulically operated.

Some embodiments of the present methods of making modified wound insertsalso comprise: cooling the foam (e.g., after heating the foam) such thatthe compressed portion of the foam remains substantially compressed atroom temperature (e.g., at a temperature of 72 degrees Fahrenheit) inthe absence of a compressive force. In other embodiments, cooling thefoam includes cooling a coating that has been applied to the foam suchthat the compressed portion remains substantially compressed in theabsence of a compressive force at a temperature or temperature rangeequal to, less than, greater than, or between any of: 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and/or 150 degreesFahrenheit.

Thick and thin regions in the foam can be formed by any suitablemethods, such as, for example, laser cutting or the like. Such methodsare taught, for example, by U.S. Patent Application Publication2011/0178451, which is incorporated herein by reference.

In such embodiments, the coating can be dispersed through the foam, suchas, for example, by spraying the foam with the coating, dipping the foamin the coating, and/or any other suitable way of dispersing the coatingin the foam. In some embodiments, for example, the foam can be coatedwith a material that has a transition temperature (e.g., melting point,glass transition, etc.) that occurs at a relatively low temperature(e.g., lower than the foam alone), or that develops stiffness as itdries. In some embodiments, the coating can be configured to enable thefoam to be compressed (and/or compression set) at lower temperatures(e.g., without heating), such that the coating becomes stiff orotherwise resistant to expansion as it cools or dries to hold the foamin its compressed configuration. For example, a fluid adhesive may beapplied to thick portions before compressing the foam and permitted todry before removing the compressive force, such that the dried adhesivewill resist expansion from the compressed thickness. In otherembodiments, the coating can be configured to compression set the foamsuch that the compression is reversible (e.g., at least partially and/orcompletely reversible) such that the foam can expand (e.g., afterplacing in or on a wound) as it warms or absorbs water. In someembodiments, the coating comprises a cross-linkable polymer and/oractivating comprises exposing the coating to light and/or elevatedtemperature (e.g., above ambient temperature, such as, for example, atemperature sufficient to cause at least part of the cross-linkablepolymer to cross-link) to cause at least some portion of thecross-linkable polymer to become modified.

Examples of suitable coatings include cross-linkable polymers thatcontain n-methylol acrylamide (NMA). NMA is a monomer that may beco-polymerized with many other monomers (such as acrylics & vinyls). Onheating, (e.g., to about 140° C.), NMA reacts with itself and otherhydroxyl-containing groups (e.g., carboxyl). Similarly, ureaformaldehyde, melamine formaldehyde, and/or phenol formaldehyde can becaused to react with themselves and other hydroxyl-containing polymersto form crosslinks. Other crosslinking agents may include, for example,modified ethylene ureas, which react with hydroxyl-containing polymersat elevated temperatures to crosslink them. Other crosslinking agentscan include peroxides which will crosslink most polymers at elevatedtemperatures. Polymers containing hydroxyl and carboxyl groups may alsobe combined, and, when heated, may form polyester crosslinks.Additionally, epoxy prepolymers can be used that have low reactivity atroom temperatures, and when heated, react quickly to form an epoxypolymer with crosslinks. Similarly, polymeric isocyanates may be usedthat will only react significantly fast at elevated temperatures and inpresence of hydroxyl groups, amines, or moisture to form polyurethanesor polyureas.

In some embodiments, a combination of high-density regions andlow-density regions cooperate to provide various characteristics for thepresent modified wound inserts. For example, the high-density regionshave a smaller aggregate cell size and increased cell density, such thatthe high-density regions have improved wicking function andmore-effectively transmit fluid (e.g., draw fluids away from the woundsurface and/or communicate fluid from a fluid source to the woundsurface more effectively than the low-density regions. The high-densityregions are generally also mechanically stronger than the low-densityregions, such that the high-density regions can provide structuralsupport for the low-density regions and/or the modified wound insert asa whole (e.g., such that the modified wound insert is resistant totearing in directions that are not parallel to the low-density regions).Additionally, the low-density regions have a larger effective cell orpore size such that the low-density regions are less-susceptible toclogging. Especially when a negative pressure is applied to draw fluidand/or exudate away from the wound and through the modified woundinsert, the larger pore size of the low-density regions may permitfluids to be drawn through the low-density regions at a higher velocitythan the fluid is drawn through the high-density regions, such thatparticulate and granular matter are drawn to and/or through thelow-density to discourage and/or decrease the likelihood of clogging inthe high-density regions. In some embodiments, the foam can also becoated with a hydrophilic material to improve wicking properties of themodified wound insert.

The low-density regions may also be configured to permit the wounddressing to bend and/or otherwise conform to a wound. For example, thelow-density regions can be relatively easier to bend (and/or lessresilient when the modified wound insert is bent or folded along alow-density region) such as to double over a modified wound insert,and/or to conform a modified wound insert to additional hardware such asplates, pins, or the like.

Typical single-density foam modified wound inserts are isotropic suchthat under negative pressure, a typical single-density foam modifiedwound insert will contract proportionally in all directions. In someembodiments, the present modified wound inserts may also be configuredto be anisotropic, such that the present modified wound inserts can beconfigured to mechanically assist with wound closure. For example,low-density regions are less-dense (and will compress more undernegative pressure) than high-density regions. As such, if negativepressure is applied to modified wound insert, low density regions willcontract more than high-density regions, such that high-density regionswill be drawn together and modified wound insert will contract laterallymore than longitudinally. In other embodiments, the present modifiedwound inserts can be configured to have alternating and sequentiallylarger closed ring-shaped high-density regions and low-density regions,such that under negative pressure, the modified wound insert willcontract laterally inward to its own center.

In some embodiments, thick portions, thin portions, high-densityregions, and/or low-density regions can be coated and/or printed (eitherbefore or after compression) to enhance the hydrophilic or hydrophobicproperties of individual regions of the foam or of the foam as a whole.Such coated regions may also contain and/or be coated with otheradditives, such as antibiotics, or blockage-reducing agents.

In some embodiments of the present invention, wound dressings comprise awound dressing configured to be positioned on a wound (e.g., 26) of apatient (e.g., 30) and/or on or in contact with the wound surface (e.g.,42).

Some embodiments of the present wound-treatment methods comprise:positioning a modified wound insert (e.g., any of the present modifiedwound inserts such as 34 a, 34 b) on a wound (e.g., 26) of a patient(e.g., 30), where the modified wound insert comprises a foam (e.g., 300)having high-density regions (e.g., 404) and low-density regions (e.g.,408) having a density that is less than the density of the high-densityregions. In some embodiments, the foam is sterile (e.g., substantiallyfree of microbes and/or bacteria). Some embodiments further comprise:coupling a drape (e.g., 38) to skin (e.g., 46) adjacent the wound suchthat the drape covers the modified wound insert and the wound, and formsa space between the drape and the wound. Some embodiments comprise:applying negative pressure to the wound through the wound dressing(e.g., through the modified wound insert). In some embodiments, applyingnegative pressure to the wound comprises activating a vacuum source(e.g., apparatus 14 of FIG. 1, or vacuum source 200 of FIG. 3) that iscoupled to the wound dressing. Some embodiments comprise: delivering afluid to the wound through the wound dressing. In some embodiments,delivering a fluid comprises activating a fluid source (e.g., fluidsource 248 of FIG. 3) that is coupled to the wound dressing.

Some embodiments of the present wound-treatment systems comprise eitherembodiment of system 10 (or any subset of components of eitherembodiment of system 10), and one or more of the present modified woundinserts and/or wound dressings.

The various illustrative embodiments of devices, systems, and methodsdescribed herein are not intended to be limited to the particular formsdisclosed. Rather, they include all modifications and alternativesfalling within the scope of the claims.

The claims are not intended to include, and should not be interpreted toinclude, means-plus- or step-plus-function limitations, unless such alimitation is explicitly recited in a given claim using the phrase(s)“means for” or “step for,” respectively.

It will be understood that the benefits and advantages described abovemay relate to one embodiment or may relate to several embodiments. Itwill further be understood that reference to ‘an’ item refers to one ormore of those items, unless otherwise specified.

The steps of the methods described herein may be carried out in anysuitable order, or simultaneously where appropriate.

Where appropriate, aspects of any of the examples described above may becombined with aspects of any of the other examples described to formfurther examples having comparable or different properties andaddressing the same or different problems.

It will be understood that the above description of preferredembodiments is given by way of example only and that variousmodifications may be made by those skilled in the art. The abovespecification, examples and data provide a complete description of thestructure and use of exemplary embodiments. Although various embodimentshave been described above with a certain degree of particularity, orwith reference to one or more individual embodiments, those skilled inthe art could make numerous alterations to the disclosed embodimentswithout departing from the scope of this invention.

E. DEVICES COMPRISING AN APTAMER-MODIFIED WOUND INSERT

The aptamer-modified polymers described above and materials madetherefrom may be used for a variety of purposes, including to (a)capture and concentrate biological targets in the wound environment,with the option to release back into the wound bed, (b) specify thechemical nature of the binding, and/or (c) dictate the orientation withwhich the target factors are presented to the cells.

A dressing or wound insert made for an aptamer-modified polymer may beused together with negative pressure wound therapy. In some embodiments,compatible foam coated with aptamers or small peptide linkers (ligands)which select for beneficial molecules in the wound fluid as it passesthrough the foam would thereby prevent their removal into the negativepressure wound therapy canister. Appropriate molecules for selectionfrom wound fluid include metabolites, growth factors, chemokines andcytokines which would not impede fluid flow through the negativepressure wound therapy dressing. In some embodiments, appropriatelinkers would bind the wound fluid molecules in such an orientation thatthe “active site” of the wound fluid molecule is still available foreliciting a biological response. For example, one such molecule may beVEGF. VEGF has specific sites on the molecule that bind to cellularreceptors. The binding of the VEGF molecule to the cellular receptor maybe used to initiate a biological response, typically angiogenesis.

In some embodiments, the methods taught herein may be used to bind tomolecules chemotactic to macrophages such as MCP1 (FIG. 15), which maybe used to stimulate macrophage migration into the wound and therebyprogress the wound from a chronic to a healing state. In someembodiments, PDGF or collagen fragments could be bound during theproliferative or late inflammatory phases to stimulate the migration offibroblasts into the wound. Stimulating the migration of macrophages andthen fibroblasts into the wound may assist in the progression of thewound through the inflammatory phase and into the proliferative phase ofwound healing. In some embodiments, Nitric Oxide Synthase could be boundfrom the wound fluid to stimulate perfusion. This may help to promotehealing by allowing a higher level of nutrients into the wound. In someembodiments, anti-inflammatory cytokines such as IL4 or IL10 could bebound to decrease inflammation, thus progressing the wound more quicklythrough the inflammatory and into the proliferative phase of healing.DNA fragments could also be bound to the dressing. In some embodiments,the binding of highly charged DNA could enable current to be passedthrough the dressing. Electrical stimulation has been used for manyyears in the treatment of wounds. Therefore, binding DNA to the wounddressing may allow for the application of current to the wound.

Experiments were conducted whether aptamers bound to a peptide (in thiscase an antibody) could still maintain biological activity. VEGF insolution was passed over beads linked to an anti-VEGF antibody. Afterpassing the solution over the beads, beads were spun down and washed.The washed beads were then used for endothelial cell migration assays.Results from this experiment showed that after 3 hours, 2 fold morecells had migrated towards the beads bound to the antibody (and VEGF)than beads with no antibody. See FIG. 18. Thus, it was shown VEGF boundto an antibody can maintain biological activity.

Referring now to the drawings, and more particularly to FIG. 1, showntherein is an embodiment of one of the present wound treatment system10. In the embodiment shown, apparatus 10 comprises a wound-treatmentapparatus 14, and a wound dressing 18 coupled to apparatus 14 by aconduit 22. As shown, dressing 18 is configured to be coupled to (and isshown coupled to) a wound 26 of a patient 30. More particularly, in theembodiment shown, dressing 18 comprises a modified wound insert 34 and adrape 38. As shown, modified wound insert 34 is configured to bepositioned (and is shown positioned) on wound 26 (e.g., on or adjacentto wound surface 42), and/or drape 38 is configured to be coupled to(and is shown coupled to) skin 46 of the patient adjacent to wound 26such that drape 38 covers modified wound insert 34 and wound 26, andforms a space 50 between drape 38 and wound 26 (e.g., wound surface 42).

Apparatus 14 can comprise, for example, a vacuum source configured to beactuatable (and/or actuated) to apply negative pressure (e.g., viaconduit 22) to wound dressing 18, a fluid source configured to beactuatable (and/or actuated) to deliver (e.g., via conduit 22) a fluid(e.g., an installation fluid such as a medicinal fluid, antibacterialfluid, irrigation fluid, and or the like) to wound dressing 18. System10 can be implemented and/or actuated and/or coupled to patient 30 inany of various configurations and/or methods similar to those describedin the prior art. For example, various wound therapy systems andcomponents are commercially available through and/or from KCI USA, Inc.of San Antonio, Tex., U.S.A., and/or its subsidiary and relatedcompanies (collectively, “KCI”).

Conduit 22 can comprise a single lumen conduit (e.g., switched between avacuum source and/or a fluid source and apparatus 14), or can comprisemultiple single-lumen conduits or a multi-lumen conduit such that, forexample, fluid can be delivered and/or negative pressure can be appliedto wound dressing 18 individually and/or simultaneously. Additionally,conduit 22 can comprise, for example, a first lumen for the applicationof negative pressure and/or fluid delivery, and at least one additionallumen for coupling to pressure sensor(s) to sense pressure or negativepressure between drape 38 and surface 42. In some embodiments, conduit22 can comprise multiple lumens (e.g., as in a single conduit with acentral lumen for application of negative pressure and/or fluiddelivery, and one or more peripheral lumens disposed adjacent or aroundthe central lumen such that the peripheral lumens can be coupled to apressure sensor to sense a pressure or negative pressure between drape38 and surface 42 (e.g. in space 50). The lumens may be arranged with acentral lumen and other lumens disposed radially around the centrallumen, or in other suitable arrangements. The lumens may also beprovided in separate conduits. In the embodiment shown, system 10further comprises a wound dressing connection pad 54 configured to becoupled (and is shown coupled) to conduit 22. One example of a suitableconnection pad 54 is the “V.A.C. T.R.A.C.® Pad,” commercially availablefrom KCI. One example of a suitable drape 38 includes the “V.A.C.®Drape” commercially available from KCI.

Referring now to FIG. 2, a side view of a modified wound insert 34 isshown. Modified wound insert 34 has an upper side 100, a lower side 104,lateral sides 108, 112 and interior volume 116. Although only one sideis shown of modified wound insert 34, it will be understood by those ofordinary skill in the art that modified wound insert 34 includes athree-dimensional rectangular volume having a depth extendingperpendicular to the side shown. In other embodiments, modified woundinsert 34 can have any suitable shape, such as, for example, a roundcylindrical shape, a fanciful shape, or may be trimmed to fit anirregular shape of a wound (e.g., 26 and/or wound surface 42). Modifiedwound insert 34 can comprise a foam, such as, for example, open-celledfoam (which may also be reticulated).

Embodiments of the present wound treatment methods may be betterunderstood with reference to FIG. 1, which depicts a schematic blockdiagram of one embodiment of system 10. In the embodiment shown, wounddressing 18 is coupled to apparatus 14, and apparatus 14 comprises avacuum source 200 (e.g., a vacuum pump and/or the like) coupled to acanister 204 (e.g., configured to receive exudate and or the like fromwound dressing 18) by way of a conduit 208. In the embodiment shown,apparatus 14 further comprises: a pressure sensor 212 having a firstpressure transducer 216 coupled to conduit 208 by way of conduit 220and/or tee-fitting 224, and a second pressure transducer 228 coupled tocanister 204 and/or wound dressing 18 by way of conduit 232. Pressuresensor 212 is configured to sense the negative pressure in wounddressing 18, and/or any of the various lumens (e.g., within conduits)coupled to wound dressing 18, pressure sensor 212, and/or vacuum source200.

In the embodiment shown, apparatus 14 further comprises a pressurerelease valve 236 coupled to conduit 232. Further, in the embodimentshown, canister 204 and vacuum source 200 are coupled to wound dressing18 by way of conduit 240; and/or canister 204 can comprise a filter 244at or near an outlet of canister 204 to prevent liquid or solidparticles from entering conduit 208. Filter 244 can comprise, forexample, a bacterial filter that is hydrophobic and/or lipophilic suchthat aqueous and/or oily liquids will bead on the surface of the filter.Apparatus 14 is typically configured such that, during operation, vacuumsource 200 will provide sufficient airflow through a filter 244 that thepressure drop across filter 244 is not substantial (e.g., such that thepressure drop will not substantially interfere with the application ofnegative pressure from wound dressing 18 from vacuum source 200).

In the embodiment shown, apparatus 14 further comprises a fluid source248 coupled to wound dressing 18 by way of a conduit 252 that is coupledto conduit 240 such as, for example, by way of a tee- or other suitablefitting 256. In some embodiments, tee fitting 256 can comprise a switchvalve and/or the like such that communication can be selectivelypermitted between wound dressing 18 and vacuum source 200, or betweenwound dressing 18 and fluid source 248. In some embodiments apparatus 14comprises only one of vacuum source 200 and fluid source 248. Inembodiments of apparatus 14 that comprise only fluid source 248,canister 204 and/or pressure sensor 212 can also be omitted. In variousembodiments, such as the one shown, conduit 232 and/or conduit 240and/or conduit 252 can be combined and/or comprised in a singlemulti-lumen conduit, such as is described above with reference toFIG. 1. In some embodiments, fluid source 248 is coupled directly towound dressing 18 (e.g., conduit 252 is coupled one end to wounddressing 18, such as via connection pad 54, and conduit 252 is coupledon the other end to fluid source 248; and conduit 252 is not coupled totee fitting 256).

In various embodiments, such as the one shown in FIG. 3, apparatus 14can be configured such that as soon as liquid in the canister reaches alevel where filter 244 is occluded, a much-increased negative (orsubatmospheric) pressure occurs in conduit 208 and is sensed bytransducer 216. Transducer 216 can be connected to circuitry thatinterprets such a pressure change as a filled canister and signals thisby means of a message on an LCD and/or buzzer that canister 204 requiresemptying and/or replacement, and/or that automatically shuts off ordisables vacuum source 200.

Apparatus 14 can also be configured to apply negative (orsubatmospheric) pressure (e.g., continuously, intermittently, and/orperiodically) to the wound site, and/or such that pressure relief valve236 enables pressure at the wound site to be brought to atmosphericpressure rapidly. Thus, if apparatus 14 is programmed, for example, torelieve pressure at ten-minute intervals, at these intervals pressurerelief valve 236 can open for a specified period, allow the pressure toequalize at the wound site, and then close to restore the negativepressure. It will be appreciated that when constant negative pressure isbeing applied to the wound site, valve 236 remains closed to preventleakage to or from the atmosphere. In this state, it is possible tomaintain negative pressure at the wound site without running and/oroperating pump 200 continuously, but only from time to time orperiodically, to maintain a desired level of negative pressure (i.e. adesired pressure below atmospheric pressure), which is sensed bytransducer 216. This saves power and enables the appliance to operatefor long periods on its battery power supply.

In some embodiments, factors may be removed, or their concentrationmodulated, using electrical pulses, light, ultrasound and temperature.

F. INSTILLATION SOLUTIONS

In some embodiments dressing made from the aptamer modified poloymersdisclosed herein may be used together with wound instillation solutions,for example in the application of a negative pressure treatment to apatient's wound. In some embodiments, the instillation solutioncomprises ingredients to help release or modulate the release of thefactors bound to the foam.

Examples of instilled ingredients which may be used in some embodimentsto dissociate bound molecules include: saline solutions, solutions withslightly acidic pH, solutions with slightly basic pH, solutions withvarious surfactants (i.e. polysorbate), solutions with slight ioniccharge, EDTA or EGTA. In some embodiments, the fluid instilled toinitiate the dissociation of the bound factors from the linker willdepend upon the binding strength of the factor-linker complex, which isin turn determined by the dissociation constant. The dissociationconstant may be modified by using knowledge of amino acid chemistry ofthe factor of interest to design the linker/peptide.

In some embodiments, the instillation solution comprises hypochlorousacid (HOCl) and hypochlorite ion. Both are examples of effectiveantimicrobial agents for biocidal action. For example, HOCl is typicallycapable of killing a broad spectrum of microbes (e.g., fungus, bacteria,viruses, fungus, yeast, and the like); often in a relatively shortperiod of time (e.g., is capable of killing greater than 99% of microbeswithin a period of less than 10 seconds). Such antimicrobial agents canbe generated or formed by a combination of the present reactive agentsand fluid (e.g., water and/or aqueous solution, such as, for example,saline solution) and may be more effective and/or more versatile thanantibiotics and other commonly used antimicrobial agents used in woundtreatment in the past. For example, antibiotics may be bacteria-specificsuch that testing may be required to determine a suitable antibiotic touse for a specific wound or infection; and/or such that antibiotics mayhave only limited effectiveness for individual wounds and/or infections(e.g., where testing is not performed and/or where a wound is infectedwith a plurality of different bacteria). Such testing may take as longas several days to determine an appropriate antibiotic, delayingtreatment or selection of an effective antibiotic. Additionally,bacteria may develop resistance to antibiotics, such that antibioticsmay have reduced effectiveness after an amount of time. Further,antibiotics are typically administered intravenously (systemically) suchthat antibiotics may kill beneficial bacteria (e.g., in a patient'sdigestive system) and/or may cause organ damage (e.g., to a patient'sliver).

In contrast, the reactive agents (and/or antimicrobial products of thereactive agents) of the present embodiments can be configured to have abroad-spectrum killing power that will kill a variety of microbes (e.g.,fungus, bacteria, viruses, fungus, yeast, etc.). Additionally, thepresent reactive agents (and/or antimicrobial products of the reactiveagents) can be delivered locally (preventing systemic damage or otherside effects to organs and the like).

However, due to the reactivity of HOCl or OCl⁻ with oxidizable organicsubstances, its utility in wound care applications has previously beenlimited. For example, prior art methods of generating hypochlorous acidhave required electrolysis of saltwater or the like (e.g., withexpensive equipment at a patient's bedside). By way of another example,commercially available chemicals (e.g., bleach) have a hypochlorous acidconcentration of 5% or greater, which is too high to permit medical uses(e.g., will cause cytoxicity). Additionally, at suitable medicalconcentrations (e.g., 2-20 mM hypochlorous acid solutions),approximately 99% or more of the solution is water, such that shippingis more expensive and/or more difficult than necessary. Further, storageof hypochlorous acid solutions is difficult, as reactions withcontainers typically degrade or reduce the concentration of thehypochlorous acid solution. However, the present wound inserts can bedeposited with reactive agents (have reactive agents deposited in thefoam of the wound inserts) such that upon application of a fluid such assaline or water, OCl (and/or ClO⁻) is released (e.g., to formhypochlorous acid) and delivered to a wound for biocidal action.

G. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Preparation and Silanation of 30 PHR Chitosan InterfacialLayer

In this procedure, a heterobifunctional silane crosslinker,3-aminopropyldiisopropylethoxysilane, was deposited on 30 per hundredresin (30 phr) chitosan foam, which was produced using 0.3 g of chitosan(Sigma Product Number 448877) per 1.0 g of an aromatic diisocyanate andtri-branched polyol mix. A plurality of the —OH groups of the copolymerwere then silylated with 3-aminopropyldiisopropylethoxysilane (Gelest™compound SIA0602.0). This was accomplished using the protocol furtherbelow.

The resulting silylated 30 phr chitosan foam was compared with silylatedreticulated open cell foam using an o-phtaldialdehyde (OPA) assay thatcompares relative fluorescence units (RFU). OPA fluoresces when it bindsto primary —NH₂ groups. Therefore, RFUs for 30 phr chitosan foam shouldbe greater than, for example, OPA alone indicating the presence primaryof —NH₂. The results from this test are shown in Table 1 below and inFIG. 5. This quantitative analysis demonstrated that the silylated 30phr chitosan foam (“30 PHR Si”) had relatively twice the number (1.25e7)of RFUs as the silylated reticulated open cell foam (ROCF Si) (6.45e6).This result is consistent with the silylated 30 phr chitosan foam havingmore free amino groups than the silylated reticulated open cell foam.

TABLE 1 OPA Assay Results of 30 PHR Si with Reticulated Open Cell Foam(“ROCF”) 30 PHR Si ROCF Si OPA 13026850 6584950 1654498 11297955 60214621685639 13362349 6735590 1701811 Mean 12,562,385 6,447,334 1,680,649 SEM1,107,803 376,428 24,048

FIG. 5 depicts results comparing the number of free amino groups ofsilylated 30 phr chitosan foam (“30 PHR Si”) with silylated reticulatedopen cell foam (“ROCF Si”) using an o-phtaldialdehyde (OPA) assay. Theresults are provided in terms of relative fluorescence units (RFU). OPAalone, which is a sensitive detection reagent for amines, also serves asthe control.

The equipment used in carrying out these experiments include:

-   -   Plate Reader (Biotek—Synergy 4)    -   TRITC capable microscope (Olympus—IX51)    -   A block of 30 phr chitosan foam    -   PBS Packs (Thermo Scientific 28384)    -   96-well, black, transparent bottom (Greiner 655209)    -   3-Aminopropyldiisopropylethoxysilane, Gelest Compound SIA0602.0        (“compound 602”). This compound was kept dessicated.    -   25 mL Glass ScintillationVials.    -   Ortho-pthaldialdehyde or OPA (Sigma P/N—P0532)    -   1 in cork boring tool    -   4 mm biopsy punch

The test procedure used included the following steps:

-   -   95% EtOH Solution was prepared.    -   15% 3-Aminopropyldiisopropylethoxysilane solution in 95% EtOH        was prepared in a fume hood in a 25 mL scintillation vial and        placed on shaker for 5 min.    -   PBS was prepared by mixing 1 pack PBS powder and 500 mL DI H₂O.    -   Sheets of 2 mm thick 30 phr foam were cut from a foam block        using a deli slicer.    -   Coupons were punched out with a 1 inch cork boring tool.    -   All sample preparation steps were performed in glass        scintillation vials.    -   Coupons were submerged in 10 mL vial of 15% silane solution and        compressed against the bottom of the vial with a glass rod to        remove any bubbles trapped inside the foam and placed on a        shaker for 15 min. Negative controls were prepared by in a        similar manner except that 10 mL of 95% EtOH solution was        substituted for the silane solution.    -   The coupons were removed from the silane solution with forceps        and transferred to a 95% EtOH bath and gently swirled        approximately 3 seconds to wash off excess silane. This wash was        then repeated.    -   The coupons were then transferred to a clean 250 mL beaker to        ensure they are not touching each other.    -   The beaker was placed for 2 hours in an oven that was preheated        to 80° C. After heating the beaker was removed from the oven and        allowed it to cool at RT. Alternatively, you the coupons may be        placed in beakers, uncovered, and allowed to incubate for 24 hr        at RT.    -   Coupons were punched from the silylated and non-silylated 30 phr        using a 4 mm biopsy punch. Each coupon was also weighed.    -   Quantification step:        -   200 μL of OPA was added to each coupon on a black 96-well            plate, the plate covered and foil and incubated for 2 min at            RT.        -   The plate was placed in the plate reader and the OPA Assay            Protocol was initiated.

Example 2 Sulfo-EMCS Conjugation of a Capture Peptide to 30 PHR ChitosanInterfacial Layer

A 30 per hundred resin (30 phr) chitosan prototype was formulated using0.3 g of chitosan (Sigma P/N 448877) to 1.0 g polyurethane. Thefollowing test apparatuses, materials and procedures were used incarrying out these experiments.

The materials used in carrying out these experiments include:

-   -   1 in. silanated chitosan coupons.    -   Sulfo-EMCS (Pierce—22307)    -   BupH Borate Buffer Packs (Pierce—28372)    -   EDTA (Sigma Aldrich—E9885-500G)    -   25 mL Scintillation Vials.

The materials used in carrying out these experiments include:

The test procedure used included the steps listed below: All buffers andreagents were prepared in advance, except the Sulfo-EMCS, which wasreconstituted immediately before use. See Sulfo-EMCS ProductInstructions (Thermo Scientific-Pierce), which is incorporated byreference herein in its entirety.

-   -   1.1. BupH Borate Buffer: Two packs BupH Borate Buffer powderwere        added to 1 L DI H₂O as per manufacturer's instructions.    -   1.2. Conjugation Buffer, BupH Borate Buffer/5 mM EDTA was        prepared by dissolving 146 mg EDTA in 500 mL BupH Borate Buffer.    -   1.3. Sulfo-EMCS, allowed to warm to RT, was reconstituted        immediately before performing the Sulfo-EMCS addition step. 50        mg Sulfo-EMCS was reconstituted in 1.22 mL BupH Borate        Buffer/EDTA for a 100× stock per manufacturer's instructions.    -   1.4. Capture peptides were reconstituted according to        manufacturer's instructions. A 10 mM working solution was        prepared.

Peptide Stock Solution: Peptide 22 mol of peptide/volume diluent = finalconc peptide concentration:  10 mM Reaction mixture: Peptide: DissolvePeptide in H2O. Desired Concentration Per Reaction: 150 uM Sulfo-EMCS:Reconstitute 41 mg ECMS in 1.22 mL BupH PBS for a 100 mM stock. Add EMCSto reaction mixture based on the # moles desired

-   -   1.5. Sample preparation steps were performed in glass        scintillation vials to minimize the amount of protein and        peptide adsorbed by the vial.    -   1.6. Overview. The experiment proceeded as follows (˜2.5 hr):        Reaction Mixture 1→Wash→Reaction Mixture 2→Wash→Used or stored        at 4° C.    -   1.7.

100× Sulfo- Reaction Mixture 1: EMCS EMCS BupH Borate Buffer/EDTA5.100E−03 Reaction Mixture 2: Peptide Peptide BupH Borate Buffer/EDTA

-   -   1.8. Reaction Mixture 1: 100× Sulfo-EMCS preparation. This        reaction bound the Sulfo-EMCS to the modified chitosan        substrate.        -   1.8.1. Preparation of Sulfo-EMCS. Add 1.22 mL of BupH Borate            Buffer/EDTA was added to a 50 mg vial of Sulfo-EMCS and then            shaken well and vortexed for ˜10 sec. Then it was allowed to            dissolve for ˜2 min, and it was used within 15 minutes of            reconstituting.        -   1.8.2. Transfer chitosan coupons from Example 1 to each vial            of reaction mixture. The negative control has no Sulfo-EMCS            or peptide; BupH Borate Buffer/EDTA was substituted for            these reagents in the reaction.        -   1.8.3. A glass rod was used to compress the silanated            chitosan coupons to ensure even reactivity with the foam            surfaces.        -   1.8.4. The reaction 1 mixture was placed in a shaker at RT            for 30 min.    -   1.9. Once the Sulfo-EMCS was conjugated, the coupons were washed        in BupH Borate Buffer. A glass rod was used to squeeze the        bubbles out of the coupons to ensure all excess Sulfo-EMCS is        removed.    -   1.10. Preparation of Reaction Mixture 2: Peptide Reaction.        -   1.10.1. The EMCS-bound foam was added to the capture peptide            (P22, a commercially available anti-GM-CSF Peptide) and BupH            Borate Buffer/EDTA mix into each reaction vial.        -   1.10.2. A glass rod was used to compress the foam coupons to            ensure even reactivity with the foam surfaces.        -   1.10.3. Reaction Mixture 2 was placed on a shaker at RT for            30 min. This conjugated the capture peptide to the            Sulfo-EMCS crosslinker deposited on the foam.    -   1.11. The coupons were then washed in BupH Borate Buffer to        remove excess peptide. Again, use a clean glass rod to remove        the bubbles to ensure even washing.    -   1.12. Use the coupons immediately for growth factor binding or        transfer the moist coupons to a capped vial and store them at        4° C. until ready for use for no longer than. 7-10 days.    -   1.13. 333 ng/mL of granulocyte macrophage colony stimulating        factor (GM-CSF) GM-CSF was incubated with the samples for 24 hr.        The unbound proteins were washed off then the samples were        eluted with an imidazole-based buffer, E1 before a buffer        exchange with PBS and quantification via ELISA. The results        indicate that the positive sample with P-22 captured 299 pg/mL        of GM-CSF, whereas the negative sample bound 124 pg/mL. See FIG.        8.

Example 3 P22 Modified Open Cell Reticulated Polymer and Capture ofCM-CSF

In this study ROCF was substituted for the chitosan substrate describedin the examples above. DyLight Fluor Texas Red (Thermo Scientific P/N46412) was used instead of OPA. It is an NH₂-reactive dye as well;however, it fluoresces at a different wavelength.

Gelest Compound 630 was used instead of Gelest Compound SIA0602.0.Compound 630 has a shorter alkane chain and no propyl groups but carriesout the same basic function.

Fluorometric Analysis of -Sil bound to ROCF. In order to determine theamount of silyl or substituted silyl groups bound to the ROCF, Texas Redfluorescent dye, an NH₂-reactive fluorophore, was conjugated to the 630linker for visualization and quantification. 630's heterobifunctionalityenables its Si atom at one end to bind to —OH groups on the foam whilethe other end presents a —NH₂ group that facilitates NH₂-reactivechemistries. Once the 630 was deposited onto the ROCF, incubation withTexas Red identified the presence of the crosslinkers on the ROCF.Fluorescent analysis showed successful deposition of silyl orsubstituted silyl groups compound 630 to ROCF (FIGS. 9A & 9B).Fluorometric quantification of the test group indicated approximately42,000 RFUs at an Ex/Em of 584/630, whereas the negative control, ROCFwithout the silyl or substituted silyl groups yielded a total of 2,586.5RFUs due to background levels of ROCF/dye interaction (FIG. 6).Therefore, -Sil conjugation resulted in a ˜16-fold increase influorescence, which corresponds to the number of -Sil linkers depositedon the foam. PBS, and ROCF in PBS were also analyzed to ensure thatautofluorescence of these materials would not skew RFU readings. Dataare given as mean±standard deviation.

With the -Sil bond, a “capture peptide” was next conjugated to theamino-functionalized ROCF. To do so, Sulfo-EMCS chemistry developed inExample 2 was used to conjugate a commercially available GM-CSFantagonist capture peptide, P22, to ROCF. The P22-linker-foam constructwas incubated with GM-CSF, washed stringently and eluted. The elutedGM-CSF was quantified via ELISA (FIG. 11). The sample with the most P22,S3, captured 49% more GM-CSF than ROCF alone. The total amount of GM-CSFcaptured by ROCF/P22, 608 pg/mL, was greater than the amount of GM-CSFbound non-specifically to the negative control, ROCF alone, 376 pg/mL.Samples S1-S3 all consisted of P22-linker-foam with Sulfo-EMCSconcentration increasing from S1 to S3.

Elution results for the P22/GM-CSF experiment: P22 was obtained from theBachem Americas Inc. 2010 peptide catalog. P22 is a GM-CSF antagonistthat binds GM-CSF in an inhibitory manner. Briefly, 330 nmol P22 wasconjugated to ROCF via Sulfo-EMCS chemistry. The P22-linker-foamconstruct was incubated with 1 μg GM-CSF overnight, washed five times,and subjected to stringent elutions. The wash preceding the elutionsshowed that non-specifically bound protein was reduced to negligiblelevels (<100 pg/mL). Upon elution, the sample with the most capturepeptide, S3, outperformed ROCF alone by 49%. The total amount of GM-CSFbound to ROCF/P22, 608 pg/mL, was greater than the amount of GM-CSFbound non-specifically to the negative control, Cl, 376 pg/mL. SamplesS1-S3 all consisted of peptide-linker-foam with Sulfo-EMCS concentrationincreasing from S1 to S3. There were no positive controls available forthis experiment, as no technology has yet been proven to covalentlyconjugate peptides to polyurethane. However, positive controls werelater developed for subsequent experiments based on experimentalresults. Data are given as mean±standard error.

Example 4 P16 Modified Polymers and Capture of VEGF

P16, an anti-VEGF peptide, was designed de novo. The design was based onthe dimeric X-ray crystallography structure of the VEGF dimer. The aminoacid residues in the true biological design can be modified to enhanceor reduce binding affinities.

P16 was designed de novo. P16 was based on the X-ray crystallographystructure of the VEGF dimer, registered as accession number 1vpf in theProtein Data Bank. The P16 peptide design mimics the natural phenomenonof dimerization that takes place between two VEGF homodimers.

P16 consists of an 11 aa sequence that mimics the dimeric interface ofChain C in Protein Data Bank structure 1vpf. The P16 sequence consistsmostly of hydrophobic residues with a few charged residues scatteredthroughout. The sole substitution is at aa1, where we substituted aglycine for a proline to minimize steric interference from the proline.

Further experiments demonstrate that P16 captures the protein targetVEGF. ELISA results from P16/VEGF experiments indicated that P16captured its protein target, VEGF (FIG. 14). P16 was conjugated to ROCF,incubated with VEGF, washed stringently, then eluted with an organicsolvent. In this experiment, the test group (P16-linker-foam) bound 584pg/mL, whereas the greatest negative control bound 321 pg/mL.Non-specific binding was expected due to the hydrophobic nature of boththe ROCF and the target protein VEGF. However, these data indicate thatstructure-based peptide design enhanced target-specific protein capturebeyond the non-specific hydrophobic properties of the ROCF.

The results summarized in both Examples 3 & 4 show that -Sil groups weresuccessfully conjugated to ROCF. Fluorometric quantification of thepositive test group indicated 21,702.5 RFUs at an Ex/Em of 584/630,whereas the negative control, ROCF without -Sil, yielded a total of2,586.5 RFUs due to background levels of ROCF/dye interaction. P16 andP22 were successfully conjugated to the linkers deposited on the ROCFsubstrate. Finally, it was possible to quantify the effectiveness of P16and P22 capturing their target proteins. In the case of P16, the elutiondata indicate that the positive sample bound 82% more VEGF than thegreatest negative control, silanated ROCF. The positive sample bound 584pg/mL, whereas the negative sample bound 321 pg/mL. For P22, the samplewith the most capture peptide outperformed ROCF alone by 49%. The totalamount of GM-CSF bound to ROCF/P22, 608 pg/mL, was greater than theamount of GM-CSF bound non-specifically to the negative control, 376pg/mL.

What is claimed is:
 1. A wound dressing comprising a polymer foam substrate conjugated to a polypeptide, wherein the polypeptide comprises a sequence at least 90% identical to SEQ ID NO:1 or a sequence at least 90% identical to SEQ ID NO:
 2. 2. The wound dressing of claim 1, wherein the polypeptide is covalently attached to the polymer foam.
 3. The wound dressing of claim 1, wherein the polypeptide is attached via one or more linkers.
 4. The wound dressing of claim 3, wherein the polypeptide is attached to the polymer foam through a thioether linkage.
 5. The wound dressing of claim 1, wherein the amino or carboxyl terminus of the polypeptide comprises a PEG spacer-cysteine residue.
 6. The wound dressing of claim 3, wherein the linker is an EMCS-derived linker or a sulfo-EMCS-derived linker.
 7. The wound dressing of claim 6, wherein the polymer foam comprises a substituted silyl derived linker.
 8. The wound dressing of claim 7, wherein the substituted silyl derived linker is derived from aminoundecyltriethoxysilane or aminopropyldiisopropylethoxysilane.
 9. The wound dressing according to claim 1, further comprising a growth factor, chemokine or cytokine bound to the wound dressing.
 10. A wound dressing comprising a polyurethane foam substrate, wherein the polyurethane foam substrate comprises a co-polymer, and wherein the co-polymer comprises a polymer polymerized with an aminoglycoside, and further comprising an aptamer covalently attached directly or via one or more linkers to a repeat unit of the co-polymer.
 11. The wound dressing of claim 10, wherein the aminoglycoside is chitosan or glucosamine.
 12. The wound dressing of claim 10, wherein the aminoglycoside is selected from the group consisting of neomycin, dibekacin, kanamycin, tobramycin, streptomycin, and gentamicin.
 13. The wound dressing of claim 10, wherein the polyurethane foam substrate is a reticulated open-celled foam.
 14. The wound dressing of claim 10, wherein the aptamer is attached to the polymer foam substrate via one or more linkers.
 15. The wound dressing of claim 14, wherein the aptamer is covalently attached to the one or more linkers through a thioether linkage.
 16. The wound dressing of claim 14, wherein the linker is an N-(e-maleimidocaproyloxy) sulfosuccinimide ester-derived linker (EMCS-derived linker) or a sulfo-EMCS-derived linker.
 17. The wound dressing of claim 14, wherein the linker is a substituted silyl-derived linker.
 18. The wound dressing of claim 17, wherein the substituted silyl-derived linker is derived from aminoundecyltriethoxysilane or aminopropyldiisopropylethoxysilane.
 19. A method for treating a wound comprising contacting a wound site with a wound dressing of claim
 1. 20. A method for treating a wound comprising contacting a wound site with a wound dressing of claim
 10. 